Derivatives of growth hormone and related proteins, and methods of use thereof

ABSTRACT

The growth hormone supergene family comprises greater than 20 structurally related cytokines and growth factors. A general method is provided for creating site-specific, biologically active conjugates of these proteins. The method involves adding cysteine residues to non-essential regions of the proteins or substituting cysteine residues for non-essential amino acids in the proteins using site-directed mutagenesis and then covalently coupling a cysteine-reactive polymer or other type of cysteine-reactive moiety to the proteins via the added cysteine residue. Disclosed herein are preferred sites for adding cysteine residues or introducing cysteine substitutions into the proteins, and the proteins and protein derivatives produced thereby. Also disclosed are therapeutic methods for using the cysteine variants of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.10/298,148, filed Nov. 15, 2002, entitled “Derivatives of Growth Hormoneand Related Proteins, and Methods of Use Thereof”, which claims thebenefit of priority under 35 U.S.C. 119(e) from U.S. ProvisionalApplication Ser. No. 60/332,285, filed Nov. 15, 2001, entitled “Methodsof Use for Granulocyte-Macrophage Colony Stimulating Factor CysteineMuteins”, and from U.S. Provisional Application Ser. No. 60/418,040,filed Oct. 11, 2002, entitled “Methods of Use for Granulocyte-MacrophageColony Stimulating Factor Cysteine Muteins.” U.S. application Ser. No.10/298,148 is also a continuation-in-part of U.S. application Ser. No.09/462,941, filed Jan. 14, 2000, entitled “Derivatives of Growth Hormoneand Related Proteins”, now U.S. Pat. No. 6,608,183, which is a nationalstage filing under 35 U.S.C. 371 of PCT Application Serial No.PCT/US98/14497, filed Jul. 13, 1998, which claims the benefit ofpriority from U.S. Provisional Application Ser. No. 60/052,516, filedJul. 14, 1997. The entire disclosure of each of the above-identifiedapplications is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to genetically engineered therapeuticproteins and methods of using such proteins. More specifically, theengineered proteins include cysteine variants of growth hormone andrelated members of the growth hormone supergene family.

BACKGROUND OF THE INVENTION

The following proteins are encoded by genes of the growth hormone (GH)supergene family (Bazan (1990); Mott and Campbell (1995); Silvennoinenand Ihle (1996); Blumberg et al. (2001)): growth hormone, prolactin,placental lactogen, erythropoietin (EPO), thrombopoietin (TPO),interleukin-2 (IL-2), IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11,IL-12 (p35 subunit), IL-13, IL-15, IL-19, IL-20, IL-21, MDA-7, IL-TIF,AK-155, oncostatin M, ciliary neurotrophic factor, leukemia inhibitoryfactor, alpha interferon, beta interferon, gamma interferon, omegainterferon, tau interferon, granulocyte-colony stimulating factor(G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF),macrophage colony stimulating factor (M-CSF) and cardiotrophin-1 (CT-1)(“the GH supergene family”). It is anticipated that additional membersof this gene family will be identified in the future through genecloning and sequencing. Members of the GH supergene family have similarsecondary and tertiary structures, despite the fact that they generallyhave limited amino acid or DNA sequence identity. The shared structuralfeatures allow new members of the gene family to be readily identified.

There is considerable interest on the part of patients and healthcareproviders in the development of long acting, “user-friendly” proteintherapeutics. Proteins are expensive to manufacture and, unlikeconventional small molecule drugs, are not readily absorbed by the body.Moreover, they are digested if taken orally. Therefore, natural proteinsmust be administered by injection. After injection, most proteins arecleared rapidly from the body, necessitating frequent, often daily,injections. Patients dislike injections, which leads to reducedcompliance and reduced drug efficacy. Some proteins, such aserythropoietin (EPO), are effective when administered less often (threetimes per week for EPO) because they are glycosylated. However,glycosylated proteins are produced using expensive mammalian cellexpression systems.

The length of time an injected protein remains in the body is finite andis determined by, e.g., the protein's size and whether or not theprotein contains covalent modifications such as glycosylation.Circulating concentrations of injected proteins change constantly, oftenby several orders of magnitude, over a 24-hour period. Rapidly changingconcentrations of protein agonists can have dramatic downstreamconsequences, at times under-stimulating and at other timesover-stimulating target cells. Similar problems plague proteinantagonists. These fluctuations can lead to decreased efficacy andincreased frequency of adverse side effects for protein therapeutics.The rapid clearance of recombinant proteins from the body significantlyincreases the amount of protein required per patient and dramaticallyincreases the cost of treatment. The cost of human proteinpharmaceuticals is expected to increase dramatically in the years aheadas new and existing drugs are approved for more disease indications.

Thus, there is a need to develop protein delivery technologies thatlower the costs of protein therapeutics to patients and healthcareproviders. The present invention provides a solution to this problem byproviding methods to prolong the circulating half-lives of proteintherapeutics in the body so that the proteins do not have to be injectedfrequently. This solution also satisfies the needs and desires ofpatients for protein therapeutics that are “user-friendly”, i.e.,protein therapeutics that do not require frequent injections. Thepresent invention solves these and other problems by providingbiologically active, cysteine-added variants of members of the growthhormone supergene family. The invention also provides for the chemicalmodification of these variants with cysteine-reactive polymers or othertypes of cysteine-reactive moieties to produce derivatives thereof andthe molecules so produced. The invention also provides for therapeuticmethods using the protein variants described herein.

SUMMARY OF THE INVENTION

The present invention provides cysteine variants of members of the GHsupergene family and uses of such cysteine variants. The variantscomprise a cysteine residue substituted for a nonessential amino acid ofthe proteins. Preferably, the variants comprise a cysteine residuesubstituted for an amino acid selected from amino acids in the loopregions, the ends of the alpha helices, proximal to the firstamphipathic helix, and distal to the final amphipathic helix or whereinthe cysteine residue is added at the N-terminus or C-terminus of theproteins. Preferred sites for substitution are the N- and O-linkedglycosylation sites. According to the present invention, “proximal tothe first amphipathic helix” refers to a position preceding, orN-terminal to, the first amphipathic helix (i.e., closer to theamino-terminus of the protein than the first amphipathic helix).Similarly, “distal to the final amphipathic helix” refers to a positionfollowing, or C-terminal to, the final amphipathic helix (i.e., furtheraway from the amino-terminus of the protein than the final amphipathichelix).

Also provided are cysteine variants wherein the amino acid substitutedfor is in the A-B loop, B-C loop, the C-D loop or D-E loop ofinterferon/interferon-10-like members of the GH supergene family.

Also provided are cysteine variants of members of the GH supergenefamily wherein the cysteine residue is introduced between two aminoacids in the natural protein. In particular, the cysteine residue isintroduced into the loop regions, the ends of the alpha helices,proximal to the first amphipathic helix, or distal to the finalamphipathic helix. Even more particularly, the cysteine variant isintroduced between two amino acids in an N—O-linked glycosylation siteor adjacent to an amino acid in an N-linked or O-linked glycosylationsite.

More particularly are provided cysteine variants wherein the loop regionwhere the cysteine is introduced is the A-B loop, the B-C loop, the C-Dloop or D-E loop of interferon/interferon-10-like members of the GHsupergene family.

Such cysteine substitutions or insertion mutations also can include theinsertion of one or more additional amino acids amino acids at theamino-terminal or carboxy-terminal to the cysteine substitution orinsertion.

Also provided are cysteine variants that are further derivatised byPEGylating the cysteine variants and including the derivatised proteinsproduced thereby.

As set forth in the examples, specific cysteine variants of the membersof the GH supergene family also are provided, including for example,variants of GH. The GH cysteine variants can have the substituted-foramino acid or inserted cysteine located at the N-terminal end of the A-Bloop, the B-C loop, the C-D loop, the first three or last three aminoacids in the A, B, C and D helices and the amino acids proximal to helixA and distal to helix D.

More particularly, the cysteine can be substituted for the followingamino acids: F1, T3, P5, E33, A34, K38, E39, Q40, S43, Q46, N47, P48,Q49, T50, S51, S55, T60, A98, N99, S100, G104, A105, S106, E129, D130,G131, S132, P133, T135, G136, Q137, K140, Q141, T142, S144, K145, D147,T148, N149, S150, H151, N152, D153, S184, E186, G187, S188, and G190.

Other examples of cysteine variants according to the invention includeerythropoietin variants. Erythropoietin variants include those whereinthe substituted for amino acid is located in the A-B loop, the B-C loop,the C-D loop, the amino acids proximal to helix A and distal to helix Dand the N- or C-terminus. Even more specifically, the EPO cysteinevariants include molecules wherein the amino acids indicated below havea cysteine substituted therefor: serine-126, N24, I25, T26, N38, I39,T40, N83, S84, A1, P2, P3, R4, D8, S9, T27, G28, A30, E31, H32, S34,N36, D43, T44, K45, N47, A50, K52, E55, G57, Q58, G77, Q78, A79, Q86,W88, E89, T107, R110, A111, G113, A114, Q115, K116, E117, A118, S120,P121, P122, D123, A124, A125, A127, A128, T132, K154, T157, G158, E159,A160, T163, G164, D165, R166 and S85.

The members of the GH supergene family include growth hormone,prolactin, placental lactogen, erythropoietin, thrombopoietin,interleukin-2, interleukin-3, interleukin-4, interleukin-5,interleukin-6, interleukin-7, interleukin-9, interleukin-10,interleukin-11, interleukin-12 (p35 subunit), interleukin-13,interleukin-15, oncostatin M, ciliary neurotrophic factor, leukemiainhibitory factor, alpha interferon, beta interferon, gamma interferon,omega interferon, tau interferon, granulocyte-colony stimulating factor,granulocyte-macrophage colony stimulating factor, macrophage colonystimulating factor, cardiotrophin-1 and other proteins identified andclassified as members of the family. The proteins can be derived fromany animal species including human, companion animals and farm animals.

Also provided are therapeutic methods for protecting an animal from adisease or condition that is amenable to treatment by any of thewild-type growth hormone superfamily proteins described herein. Themethods include administration of one or more cysteine variants(cysteine muteins) of the invention (including those with agonist orantagonist functions) to an animal for treatment of a disease orcondition.

In one aspect, provided is a method to protect an animal from a diseaseor condition, comprising administering to the animal a compositioncomprising a granulocyte-macrophage colony stimulating factor (GM-CSF)cysteine mutein as described herein.

Also provided is a method to prevent or treat the occurrence ofneutropenia in an animal comprising administering to the animal acomposition comprising a granulocyte-macrophage colony stimulatingfactor (GM-CSF) cysteine mutein as described herein. The neutropenia tobe prevented or treated using this method can include, but is notlimited to: (a) neutropenia resulting from myelosuppressivechemotherapy; (b) neutropenia associated with bone marrowtransplantation (c) neutropenia associated with infection with the humanimmunodeficiency virus; (d) neutropenia associated with burns, surgery,dilatation, anemia and neonatal septicemia; (e) severe chronicneutropenia; or neutropenia associated with aplastic anemia and acuteleukemia.

Also provided is a method to protect an animal from a disease orcondition by stimulating proliferation and differentiation ofhematopoietic cells in the animal, comprising administering to theanimal a composition comprising a granulocyte-macrophage colonystimulating factor (GM-CSF) cysteine mutein as described herein.

Also provided is a method for stimulating the expansion andproliferation of peripheral blood progenitor cells in an animalcomprising administering to the animal a composition comprising agranulocyte-macrophage colony stimulating factor (GM-CSF) cysteinemutein as described herein.

Other variations and modifications to the invention will be obvious tothose skilled in the art based on the specification and the “rules” setforth herein. All of these are considered as part of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to cysteine variants and, among otherthings, the site-specific conjugation of such proteins with polyethyleneglycol (PEG) or other such moieties. PEG is a non-antigenic, inertpolymer that significantly prolongs the length of time a proteincirculates in the body. This allows the protein to be effective for alonger period of time. Covalent modification of proteins with PEG hasproven to be a useful method to extend the circulating half-lives ofproteins in the body (Abuchowski et al., 1984; Hershfield, 1987; Meyerset al., 1991). Covalent attachment of PEG to a protein increases theprotein's effective size and reduces its rate of clearance rate from thebody. PEGs are commercially available in several sizes, allowing thecirculating half-lives of PEG-modified proteins to be tailored forindividual indications through use of different size PEGs. Otherbenefits of PEG modification include an increase in protein solubility,an increase in vivo protein stability and a decrease in proteinimmunogenicity (Katre et al., 1987; Katre, 1990).

The preferred method for PEGylating proteins is to covalently attach PEGto cysteine residues using cysteine-reactive PEGs. A number of highlyspecific, cysteine-reactive PEGs with different reactive groups (e.g.,maleimide, vinylsulfone) and different size PEGs (2-20 kDa) arecommercially available (e.g., from Shearwater, Polymers, Inc.,Huntsville, Ala.). At neutral pH, these PEG reagents selectively attachto “free” cysteine residues, i.e., cysteine residues not involved indisulfide bonds. The conjugates are hydrolytically stable. Use ofcysteine-reactive PEGs allows the development of homogeneous PEG-proteinconjugates of defined structure.

Considerable progress has been made in recent years in determining thestructures of commercially important protein therapeutics andunderstanding how they interact with their protein targets, e.g.,cell-surface receptors, proteases, etc. This structural information canbe used to design PEG-protein conjugates using cysteine-reactive PEGs.Cysteine residues in most proteins participate in disulfide bonds andare not available for PEGylation using cysteine-reactive PEGs. Throughin vitro mutagenesis using recombinant DNA techniques, additionalcysteine residues can be introduced anywhere into the protein. The addedcysteines can be introduced at the beginning of the protein, at the endof the protein, between two amino acids in the protein sequence or,preferably, substituted for an existing amino acid in the proteinsequence. The newly added “free” cysteines can serve as sites for thespecific attachment of a PEG molecule using cysteine-reactive PEGs. Theadded cysteine must be exposed on the protein's surface and accessiblefor PEGylation for this method to be successful. If the site used tointroduce an added cysteine site is non-essential for biologicalactivity, then the PEGylated protein will display essentially wild type(normal) in vitro bioactivity. The major technical challenge inPEGylating proteins with cysteine-reactive PEGs is the identification ofsurface exposed, non-essential regions in the target protein wherecysteine residues can be added or substituted for existing amino acidswithout loss of bioactivity.

Cysteine-added variants of a few human proteins and PEG-polymerconjugates of these proteins have been described. U.S. Pat. No.5,206,344 describes cysteine-added variants of IL-2. Thesecysteine-added variants are located within the first 20 amino acids fromthe amino terminus of the mature IL-2 polypeptide chain. The preferredcysteine variant is at position 3 of the mature polypeptide chain, whichcorresponds to a threonine residue that is O-glycosylated in thenaturally occurring protein. Substitution of cysteine for threonine atposition 3 yields an IL-2 variant that can be PEGylated with acysteine-reactive PEG and retain full in vitro bioactivity (Goodson andKatre, 1990). In contrast, natural IL-2 PEGylated with lysine-reactivePEGs displays reduced in vitro bioactivity (Goodson and Katre, 1990).The effects of cysteine substitutions at other positions in IL-2 werenot reported.

U.S. Pat. No. 5,166,322 teaches cysteine-added variants of IL-3. Thesevariants are located within the first 14 amino acids from the N-terminusof the mature protein sequence. The patent teaches expression of theproteins in bacteria and covalent modification of the proteins withcysteine-reactive PEGs. No information is provided as to whether thecysteine-added variants and PEG-conjugates of IL-3 are biologicallyactive. Cysteine-added variants at other positions in the polypeptidechain were not reported.

PCT Publication No. WO 9412219 and PCT Application No. PCT/US95/06540teach cysteine-added variants of insulin-like growth factor-I (IGF-I).IGF-I has a very different structure from GH and is not a member of theGH supergene family (Mott and Campbell, 1995). Cysteine substitutions atmany positions in the IGF-I protein are described. Only certain of thecysteine-added variants are biologically active. The preferred site forthe cysteine added variant is at amino acid position 69 in the matureprotein chain. Cysteine substitutions at positions near the N-terminusof the protein (residues 1-3) yielded IGF-I variants with reducedbiological activities and improper disulfide bonds.

PCT Publication No. WO 9422466 teaches two cysteine-added variants ofinsulin-like growth factor (IGF) binding protein-1, which has a verydifferent structure than GH and is not a member of the GH supergenefamily. The two cysteine-added IGF binding protein-1 variants disclosedare located at positions 98 and 101 in the mature protein chain andcorrespond to serine residues that are phosphorylated in thenaturally-occurring protein.

U.S. patent application Ser. No. 07/822,296 teaches cysteine addedvariants of tumor necrosis factor binding protein, which is a soluble,truncated form of the tumor necrosis factor cellular receptor. Tumornecrosis factor binding protein has a very different structure than GHand is not a member of the GH supergene family.

IGF-I, IGF binding protein-1 and tumor necrosis factor binding proteinhave secondary and tertiary structures that are very different from GHand the proteins are not members of the GH supergene family. Because ofthis, it is difficult to use the information gained from studies ofIGF-I, IGF binding protein-1 and tumor necrosis factor binding proteinto create cysteine-added variants of members of the GH supergene family.The studies with IL-2 and IL-3 were carried out before the structures ofIL-2 and IL-3 were known (McKay 1992; Bazan, 1992) and before it wasknown that these proteins are members of the GH supergene family.Previous experiments aimed at identifying preferred sites for addingcysteine residues to IL-2 and IL-3 were largely empirical and wereperformed prior to experiments indicating that members of the GHsupergene family possessed similar secondary and tertiary structures.

Based on the structural information now available for members of the GHsupergene family, the present invention provides “rules” for determininga priori which regions and amino acid residues in members of the GHsupergene family can be used to introduce or substitute cysteineresidues without significant loss of biological activity. In contrast tothe naturally occurring proteins, these cysteine-added variants ofmembers of the GH supergene family will possess novel properties such asthe ability to be covalently modified at defined sites within thepolypeptide chain with cysteine-reactive polymers or other types ofcysteine-reactive moieties. The covalently modified proteins will bebiologically active.

GH is the best-studied member of the GH supergene family. GH is a 22 kDaprotein secreted by the pituitary gland. GH stimulates metabolism ofbone, cartilage and muscle and is the body's primary hormone forstimulating somatic growth during childhood. Recombinant human GH (rhGH)is used to treat short stature resulting from GH inadequacy and renalfailure in children. GH is not glycosylated and can be produced in afully active form in bacteria. The protein has a short in vivo half-lifeand must be administered by daily subcutaneous injection for maximumeffectiveness (MacGillivray et al., 1996). Recombinant human GH (rhGH)was approved recently for treating cachexia in AIDS patients and isunder study for treating cachexia associated with other diseases.

The sequence of human GH is well known (see, e.g., Martial et al. 1979;Goeddel et al. 1979 which are incorporated herein by reference; SEQ IDNO:1). GH is closely related in sequence to prolactin and placentallactogen and these three proteins were considered originally to comprisea small gene family. The primary sequence of GH is highly conservedamong animal species (Abdel-Meguid et al., 1987), consistent with theprotein's broad species cross-reactivity. The three dimensional foldingpattern of porcine GH has been solved by X-ray crystallography(Abdel-Meguid et al., 1987). The protein has a compact globularstructure, comprising four amphipathic alpha helical bundles joined byloops. Human GH has a similar structure (de Vos et al., 1992). The fouralpha helical regions are termed A-D beginning from the N-terminus ofthe protein. The loop regions are referred to by the helical regionsthey join, e.g., the A-B loop joins helical bundles A and B. The A-B andC-D loops are long, whereas the B-C loop is short. GH contains fourcysteine residues, all of which participate in disulfide bonds. Thedisulfide assignments are cysteine53 joined to cysteine165 andcysteine182 joined to cysteine189.

The crystal structure of GH bound to its receptor revealed that GH hastwo receptor binding sites and binds two receptor molecules (Cunninghamet al., 1991; de Vos et al., 1992). The two receptor binding sites arereferred to as site I and site II. Site I encompasses the Carboxy(C)-terminal end of helix D and parts of helix A and the A-B loop,whereas site II encompasses the Amino (N)-terminal region of helix A anda portion of helix C. Binding of GH to its receptor occurs sequentially,with site I always binding first. Site II then engages a second GHreceptor, resulting in receptor dimerization and activation of theintracellular signaling pathways that lead to cellular responses to GH.A GH mutein in which site II has been mutated (a glycine to argininemutation at amino acid 120) is able to bind a single GH receptor, but isunable to dimerize GH receptors; this mutein acts as a GH antagonist invitro, presumably by occupying GH receptor sites without activatingintracellular signaling pathways (Fuh et al., 1992).

The roles of particular regions and amino acids in GH receptor bindingand intracellular signaling also have been studied using techniques suchas mutagenesis, monoclonal antibodies and proteolytic digestion. Thefirst mutagenesis experiments entailed replacing entire domains of GHwith similar regions of the closely related protein, prolactin(Cunningham et al., 1989). One finding was that replacement of the B-Cloop of GH with that of prolactin did not affect binding of the hybridGH protein to a soluble form of the human GH receptor, implying that theB-C loop was non-essential for receptor binding. Alanine scanningmutagenesis (replacement of individual amino acids with alanine)identified 14 amino acids that are critical for GH bioactivity(Cunningham and Wells, 1989). These amino acids are located in thehelices A, B, C, and D and the A-B loop and correspond to sites I and IIidentified from the structural studies. Two lysine residues at aminoacid positions 41 and 172, K41 and K172, were determined to be criticalcomponents of the site I receptor binding site, which explains thedecrease in bioactivity observed when K172 is acetylated (Teh andChapman, 1988). Modification of K168 also significantly reduced GHreceptor binding and bioactivity (de la Llosa et al., 1985; Martal etal., 1985; Teh and Chapman, 1988). Regions of GH responsible for bindingthe GH receptor have also been studied using monoclonal antibodies(Cunningham et al., 1989). A series of eight monoclonal antibodies wasgenerated to human GH and analyzed for the ability to neutralize GHactivity and prevent binding of GH to its recombinant soluble receptor.The latter studies allowed the putative binding site for each monoclonalantibody to be localized within the GH three-dimensional structure. Ofinterest was that monoclonal antibodies 1 and 8 were unable to displaceGH from binding its receptor. The binding sites for these monoclonalantibodies were localized to the B-C loop (monoclonal number 1) and theN-terminal end of the A-B loop (monoclonal number 8). No monoclonalswere studied that bound the C-D loop specifically. The monoclonalantibody studies suggest that the B-C loop and N-terminal end of the A-Bloop are non-essential for receptor binding. Finally, limited cleavageof GH with trypsin was found to produce a two chain derivative thatretained full activity (Mills et al., 1980; Li, 1982). Mapping studiesindicated that trypsin cleaved and/or deleted amino acids betweenpositions 134 and 149, which corresponds to the C-D loop. These studiessuggest the C-D loop is not involved in receptor binding or GHbioactivity.

Structures of a number of cytokines, including G-CSF (Hill et al.,1993), GM-CSF (Diederichs et al., 1991; Walter et al., 1992), IL-2(Bazan, 1992; McKay, 1992), IL-4 (Redfield et al., 1991; Powers et al.,1992), and IL-5 (Milburn et al., 1993) have been determined by X-raydiffraction and NMR studies and show striking conservation with the GHstructure, despite a lack of significant primary sequence homology. EPOis considered to be a member of this family based upon modeling andmutagenesis studies (Boissel et al., 1993; Wen et al., 1994). A largenumber of additional cytokines and growth factors including ciliaryneurotrophic factor (CNTF), leukemia inhibitory factor (LIF),thrombopoietin (TPO), oncostatin M, macrophage colony stimulating factor(M-CSF), IL-3, IL-6, IL-7, IL-9, IL-12, IL-13, IL-15, and alpha, beta,omega, tau and gamma interferon belong to this family (reviewed in Mottand Campbell, 1995; Silvennoinen and Ihle 1996). All of the abovecytokines and growth factors are now considered to comprise one largegene family, of which GH is the prototype.

In addition to sharing similar secondary and tertiary structures,members of this family share the property that they must oligomerizecell surface receptors to activate intracellular signaling pathways.Some GH family members, e.g., GH and EPO, bind a single type of receptorand cause it to form homodimers. Other family members, e.g., IL-2, IL-4,and IL-6, bind more than one type of receptor and cause the receptors toform heterodimers or higher order aggregates (Davis et al., 1993;Paonessa et al., 1995; Mott and Campbell, 1995). Mutagenesis studieshave shown that, like GH, these other cytokines and growth factorscontain multiple receptor binding sites, typically two, and bind theircognate receptors sequentially (Mott and Campbell, 1995; Matthews etal., 1996). Like GH, the primary receptor binding sites for these otherfamily members occur primarily in the four alpha helices and the A-Bloop (reviewed in Mott and Campbell, 1995). The specific amino acids inthe helical bundles that participate in receptor binding differ amongstthe family members (Mott and Campbell, 1995). Most of the cell surfacereceptors that interact with members of the GH supergene family arestructurally related and comprise a second large multi-gene family(Bazan, 1990; Mott and Campbell, 1995; Silvennoinen and Ihle 1996).

A general conclusion reached from mutational studies of various membersof the GH supergene family is that the loops joining the alpha helicesgenerally tend to not be involved in receptor binding. In particular theshort B-C loop appears to be non-essential for receptor binding in most,if not all, family members. For this reason, the B-C loop is a preferredregion for introducing cysteine substitutions in members of the GHsupergene family. The A-B loop, the B-C loop, the C-D loop (and D-E loopof interferon/IL-10-like members of the GH superfamily) also arepreferred sites for introducing cysteine mutations. Amino acids proximalto helix A and distal to the final helix also tend not to be involved inreceptor binding and also are preferred sites for introducing cysteinesubstitutions. Certain members of the GH family, e.g., EPO, IL-2, IL-3,IL-4, IL-6, G-CSF, GM-CSF, TPO, IL-10, IL-12 p35, IL-13, IL-15 andbeta-interferon contain N-linked and O-linked sugars. The glycosylationsites in the proteins occur almost exclusively in the loop regions andnot in the alpha helical bundles. Because the loop regions generally arenot involved in receptor binding and because they are sites for thecovalent attachment of sugar groups, they are preferred sites forintroducing cysteine substitutions into the proteins. Amino acids thatcomprise the N- and O-linked glycosylation sites in the proteins arepreferred sites for cysteine substitutions because these amino acids aresurface-exposed, the natural protein can tolerate bulky sugar groupsattached to the proteins at these sites and the glycosylation sites tendto be located away from the receptor binding sites.

Many additional members of the GH gene family are likely to bediscovered in the future. New members of the GH supergene family can beidentified through computer-aided secondary and tertiary structureanalyses of the predicted protein sequences. Members of the GH supergenefamily will possess four or five amphipathic helices joined bynon-helical amino acids (the loop regions). The proteins may contain ahydrophobic signal sequence at their N-terminus to promote secretionfrom the cell. Such later discovered members of the GH supergene familyalso are included within this invention.

The present invention provides “rules” for creating biologically activecysteine-added variants of members of the GH supergene family. These“rules” can be applied to any existing or future member of the GHsupergene family. The cysteine-added variants will posses novelproperties not shared by the naturally occurring proteins. Mostimportantly, the cysteine added variants will possess the property thatthey can be covalently modified with cysteine-reactive polymers or othertypes of cysteine-reactive moieties to generate biologically activeproteins with improved properties such as increased in vivo half-life,increased solubility and improved in vivo efficacy.

Specifically, the present invention provides biologically activecysteine variants of members of the GH supergene family by substitutingcysteine residues for non-essential amino acids in the proteins.Preferably, the cysteine residues are substituted for amino acids thatcomprise the loop regions, for amino acids near the ends of the alphahelices and for amino acids proximal to the first amphipathic helix ordistal to the final amphipathic helix of these proteins. Other preferredsites for adding cysteine residues are at the N-terminus or C-terminusof the proteins. Cysteine residues also can be introduced between twoamino acids in the disclosed regions of the polypeptide chain. Thepresent invention teaches that N- and O-linked glycosylation sites inthe proteins are preferred sites for introducing cysteine substitutionseither by substitution for amino acids that make up the sites or, in thecase of N-linked sites, introduction of cysteines therein. Theglycosylation sites can be serine or threonine residues that areO-glycosylated or asparagine residues that are N-glycosylated. N-linkedglycosylation sites have the general structure asparagine-X-serine orthreonine (N—X—S/T), where X can be any amino acid. The asparagineresidue, the amino acid in the X position and the serine/threonineresidue of the N-linked glycosylation site are preferred sites forcreating biologically active cysteine-added variants of these proteins.Amino acids immediately surrounding or adjacent to the O-linked andN-linked glycosylation sites (within about 10 residues on either side ofthe glycosylation site) are preferred sites for introducingcysteine-substitutions.

More generally, certain of the “rules” for identifying preferred sitesfor creating biologically active cysteine-added protein variants can beapplied to any protein, not just proteins that are members of the GHsupergene family. Specifically, preferred sites for creatingbiologically active cysteine variants of proteins (other than IL-2) areO-linked glycosylation sites. Amino acids immediately surrounding theO-linked glycosylation site (within about 10 residues on either side ofthe glycosylation site) also are preferred sites. N-linked glycosylationsites, and the amino acid residues immediately adjacent on either sideof the glycosylation site (within about 10 residues of the N—X—S/T site)also are preferred sites for creating cysteine added protein variants.Amino acids that can be replaced with cysteine without significant lossof biological activity also are preferred sites for creatingcysteine-added protein variants. Such non-essential amino acids can beidentified by performing cysteine-scanning mutagenesis on the targetprotein and measuring effects on biological activity. Cysteine-scanningmutagenesis entails adding or substituting cysteine residues forindividual amino acids in the polypeptide chain and determining theeffect of the cysteine substitution on biological activity. Cysteinescanning mutagenesis is similar to alanine-scanning mutagenesis(Cunningham et al., 1992), except that target amino acids areindividually replaced with cysteine rather than alanine residues.

Application of the “rules” to create cysteine-added variants andconjugates of protein antagonists also is contemplated. Excessproduction of cytokines and growth factors has been implicated in thepathology of many inflammatory conditions such as rheumatoid arthritis,asthma, allergies and wound scarring. Excess production of GH has beenimplicated as a cause of acromegaly. Certain growth factors andcytokines, e.g., GH and IL-6, have been implicated in proliferation ofparticular cancers. Many of the growth factors and cytokines implicatedin inflammation and cancer are members of the GH supergene family. Thereis considerable interest in developing protein antagonists of thesemolecules to treat these diseases. One strategy involves engineering thecytokines and growth factors so that they can bind to, but notoligomerize receptors. This is accomplished by mutagenizing the secondreceptor binding site (site II) on the molecules. The resulting muteinsare able to bind and occupy receptor sites but are incapable ofactivating intracellular signaling pathways. This strategy has beensuccessfully applied to GH to make a GH antagonist (Cunningham et al.,1992). Similar strategies are being pursued to develop antagonists ofother members of the GH supergene family such as IL-2 (Zurawski et al.,1990; Zurawski and Zurawski, 1992), IL-4 (Kruse et al., 1992), IL-5(Tavernier et al., 1995), GM-CSF (Hercus et al., 1994) and EPO (Matthewset al., 1996). Since the preferred sites for adding cysteine residues tomembers of the GH supergene family described here lie outside of thereceptor binding sites in these proteins, and thus removed from anysites used to create protein antagonists, the cysteine-added variantsdescribed herein could be used to generate long-acting versions ofprotein antagonists. As an example, Cunningham et al. (1992) developedan in vitro GH antagonist by mutating a glycine residue (amino acid 120)to an arginine. This glycine residue is a critical component of thesecond receptor binding site in GH; when it is replaced with arginine,GH cannot dimerize receptors. The glycine to arginine mutation atposition 120 can be introduced into DNA sequences encoding thecysteine-added variants of GH contemplated herein to create acysteine-added GH antagonist that can be conjugated withcysteine-reactive PEGs or other types of cysteine-reactive moieties.Similarly, amino acid changes in other proteins that turn the proteinsfrom agonists to antagonists could be incorporated into DNA sequencesencoding cysteine-added protein variants described herein. Considerableeffort is being spent to identify amino acid changes that convertprotein agonists to antagonists. Hercus et al. (1994) reported thatsubstituting arginine or lysine for glutamic acid at position 21 in themature GM-CSF protein converts GM-CSF from an agonist to an antagonist.Tavernier et al. (1995) reported that substituting glutamine forglutamic acid at position 13 of mature IL-5 creates an IL-5 antagonist.Prolactin antagonists can be created by substituting other amino acids,in particular alanine, aspartic acid and glutamic acid, for serine-179of prolactin (Chen et al., 1998). Receptor antagonists of erythropoietincan be created by substituting alanine for arginine-103 oferythropoietin (Matthews et al., 1996).

Experimental strategies similar to those described above can be used tocreate cysteine-added variants (both agonists and antagonists) ofmembers of the GH supergene family derived from various animals. This ispossible because the primary amino acid sequences and structures ofcytokines and growth factors are largely conserved between human andanimal species. For this reason, the “rules” disclosed herein forcreating biologically active cysteine-added variants of members of theGH supergene family will be useful for creating biologically activecysteine-added variants of members of the GH supergene family ofcompanion animals (e.g., dogs, cats, horses) and commercial animal(e.g., cow, sheep, pig) species. Conjugation of these cysteine-addedvariants with cysteine-reactive PEGs will create long-acting versions ofthese proteins that will benefit the companion animal and commercialfarm animal markets.

Proteins that are members of the GH supergene family (hematopoieticcytokines) are provided in Silvennoimem and Ihle (1996). Silvennoimemand Ihle (1996) also provide information about the structure andexpression of these proteins. DNA sequences, encoded amino acids and invitro and in vivo bioassays for the proteins described herein aredescribed in Aggarwal and Gutterman (1992; 1996), Aggarwal (1998), andSilvennoimem and Ihle (1996). Bioassays for the proteins also areprovided in catalogues of various commercial suppliers of these proteinssuch as R&D Systems, Inc. and Endogen, Inc.

The cysteine variants of the present invention can be used for any ofthe known therapeutic uses of the native proteins in essentially thesame forms and doses all well known in the art. By way of example,therapeutic methods for increasing hematopoiesis in a patient and foraccelerating recovery from neutropenia are described herein which use acysteine variant of granulocyte macrophage colony stimulating factor(GM-CSF) according to the present invention. It is to be understood,however, that general discussion regarding modes of administration,dosage and delivery of cysteine variants such as the GM-CSF cysteinemutein, is generally intended to apply to therapeutic methods using anyof the cysteine variants described herein.

One embodiment of the invention relates to a method to protect an animalfrom a disease or condition, comprising administering to the animal acomposition comprising a granulocyte-macrophage colony stimulatingfactor (GM-CSF) cysteine mutein as described herein. In one embodiment,the cysteine mutein is prepared using methods described in PCTApplication No. PCT/US01/16088 (PCT Publication No. WO 01/87925 A2),incorporated herein by reference in its entirety.

Another embodiment of the invention relates to a method to prevent ortreat the occurrence of neutropenia in an animal comprisingadministering to the animal a composition comprising agranulocyte-macrophage colony stimulating factor (GM-CSF) cysteinemutein as described herein and in one embodiment, as prepared usingmethods described in PCT Application No. PCT/US01/16088, supra. Theneutropenia to be prevented or treated using this method can include,but is not limited to: (a) neutropenia resulting from myelosuppressivechemotherapy; (b) neutropenia associated with bone marrowtransplantation (c) neutropenia associated with infection with the humanimmunodeficiency virus; (d) neutropenia associated with burns, surgery,dilatation, anemia and neonatal septicemia; (e) severe chronicneutropenia; or neutropenia associated with aplastic anemia and acuteleukemia.

Another embodiment of the present invention relates to a method toprotect an animal from a disease or condition by stimulatingproliferation and differentiation of hematopoietic cells in the animal,comprising administering to said animal a composition comprising agranulocyte-macrophage colony stimulating factor (GM-CSF) cysteinemutein as described herein and in one embodiment, as prepared usingmethods described in PCT Application No. PCT/US01/16088, supra.Hematopoeitic cells include, but are not limited to, neutrophil,monocyte, eosinophil, erythroid, and megakaryocyte cell lineages.According to the present invention, a cell of hematopoietic lineage isable to develop into cell types including, but not limited to,erythrocyte cells (i.e. a red blood cell), leukocyte cells (i.e. a whiteblood cell), or thrombocyte cells (i.e. platelet cell). Leukocyte cellsinclude, but are not limited to, granular leukocytes, includingeosinophils, basophils, neutrophils, and mast cells; as well asnon-granular leukocytes, including megakaryocytes, polymorphonuclearcells, lymphocytes and monocytes (i.e. macrophages). According to thepresent invention, the term “lineage” refers to all of the stages of thedevelopment of a cell type, from the earliest precursor cell to acompletely mature cell (i.e. a specialized cell). As used herein, theterms “develop”, “differentiate” and “mature” all refer to theprogression of a cell from the stage of having the potential todifferentiate into at least two different cellular lineages to becominga specialized cell. Such terms can be used interchangeably for thepurposes of the present application.

Yet another embodiment of the present invention relates to a method forstimulating the expansion and proliferation of peripheral bloodprogenitor cells in an animal comprising administering to the animal acomposition comprising a granulocyte-macrophage colony stimulatingfactor (GM-CSF) cysteine mutein as described herein and in oneembodiment, as prepared using methods described in PCT Application No.PCT/US01/16088, supra. Reference to a progenitor cell or a precursorcell is reference to a group of cells capable of developing into a moremature cell. A precursor cell population can comprise cells that aretotipotent, cells that are pluripotent and cells that are stem celllineage restricted (i.e. cells capable of developing into less than allhematopoietic lineages, or into, for example, only cells of erythroidlineage). Peripheral blood progenitor cells are cells that differentiateinto various cells that circulate in the blood, such as peripheral bloodmononuclear cells (PBMC).

Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) is aglycoprotein having a molecular mass of 14.5-35 kDa that regulates theproliferation and differentiation of hematopoietic progenitor cells intomature cells such as mature neutrophils, macrophages and eosinophils.GM-CSF also stimulates the functional properties of mature monocytes,neutrophils and eosinophils. Recombinant GM-CSF is used to ameliorateneutropenia following myelosuppressive chemotherapy and bone marrowtransplantation. GM-CSF also has been used to treat severe chronicneutropenia, aplastic anemia, and acute leukemia, and to mobilizeperipheral blood progenitor cells for transplantation and blood banking.GM-CSF also may be useful for reversing neutropenia associated with thehuman immunodeficiency virus, burns, surgery, dilatation, anemia andneonatal septicemia. The ability of GM-CSF to enhance the functionalproperties of monocytes, neutrophils and eosinophils suggests thatGM-CSF may have utility as an anti-neoplastic agent either alone or incombination with other anti-neoplastic agents such as cytotoxic drugs,chemotherapeutic agents, polyclonal antibodies and monoclonal antibodiestargeting tumor cells. The immunomodulatory properties of GM-CSF alsosuggest that GM-CSF may have utility as an adjuvant for vaccines againsttumors and infectious agents.

GM-CSF has a short circulating half life, which necessitates dailysubcutaneous injections for maximum effectiveness in humans. The presentinventors have created novel GM-CSF analogs (i.e., the GM-CSF cysteinemuteins described herein) with improved in vivo characteristics such asincreased circulating half-life and improved therapeutic efficacythrough site-specific chemical modification of the protein withcysteine-reactive Polyethylene Glycol (PEG) reagents. These analogs werecreated by introducing a “free” cysteine residue (i.e., a cysteineresidue not involved in a disulfide bond) into the protein usingsite-directed mutagenesis. The free cysteine residue serves as the sitefor covalent modification of the protein with cysteine-reactive PEGreagents. The present application teaches a variety of human GM-CSFcysteine muteins that can be modified with cysteine-reactive PEGreagents and retain biological activity, and PCT Application No.PCT/US01/16088, supra, describes additional methods of preparing suchmuteins.

Human and rodent GM-CSF proteins perform similar functions in theirrespective species and studies with rodent GM-CSF proteins can be usedto predict the function of human GM-CSF in humans. Human and rodentGM-CSF proteins share 50-60% amino acid identity, but there is no crossspecies cross-reactivity in terms of biological activity or receptorbinding. It is possible to use the significant amino acid identitybetween human and rodent GM-CSF proteins to construct murine GM-CSFcysteine muteins that are analogues of human GM-CSF cysteine muteins.The murine GM-CSF cysteine analogs can be expressed, purified andPEGylated using procedures similar to those described for human GM-CSFcysteine variants herein and in PCT Application No. PCT/US01/16088,supra. Biological activities of the PEGylated murine GM-CSF cysteinemuteins can be tested in rodent animal disease models and used topredict the effectiveness of PEGylated human GM-CSF cysteine analogs inhumans. Toward that end, the present inventors constructed the murineGM-CSF T3C mutein, which is an analog of the human GM-CSF A3C muteindescribed herein. As shown in Example 26, the PEGylated murine T3Canalog is effective at stimulating hematopoiesis in a mammal (a rodent),which indicates that PEGylated human GM-CSF cysteine muteins will beeffective at stimulating hematopoiesis in humans. Stimulatinghematopoiesis will be useful for ameliorating disease indications inwhich hematopoiesis is impaired, such as neutropenia (see Example 27).PEGylated human GM-CSF cysteine analogs can used to ameliorateneutropenia following myelosuppressive chemotherapy and bone marrowtransplantation based on the observed efficacy of GM-CSF in theseindications (Cebon and Lieschke 1994). Similarly PEGylated human GM-CSFcysteine analogs will be useful in treating diseases and conditions forwhich the therapeutic use of GM-CSF is well established (Armitage, 1998)such as peripheral blood cell progenitor cell transplantation,engraftment failure or delay after bone marrow transplantation, andinduction therapy for acute myelogenous leukemia.

Similarly, based on the observed effects of GM-CSF, PEGylated humanGM-CSF cysteine analogs also may be useful in treatment of a widevariety of cancers such as breast cancer, lung cancer, non-Hodgkin'slymphoma and ovarian cancer, by allowing chemotherapy doseintensification (Cebon and Lieschke 1994). Human GM-CSF also can be usedto treat severe chronic neutropenia, for example in Felty syndrome(Joseph et al., 1991) or myelokathexis (Hess et al.), as well asaplastic anemia, and acute leukemia. Human GM-CSF (and therefore GM-CSFcysteine variants of the invention) also may be useful for reversingneutropenia associated with the human immunodeficiency virus infection,myelodysplastic syndrome and idiopathic neutropenia (Kaczmarski et al.,1993). Neutropenia resulting from other causes such as burns, surgery,dilatation, anemia and neonatal septicemia may also be treated byadministration of PEGylated human GM-CSF cysteine analogs.

The ability of GM-CSF to enhance the functional properties of monocytes,neutrophils and eosinophils suggests that PEGylated human GM-CSFcysteine analogs may have utility as an anti-neoplastic agent eitheralone or in combination with other anti-neoplastic agents such ascytotoxic drugs, chemotherapeutic agents, polyclonal antibodies andmonoclonal antibodies targeting tumor cells (Armitage, 1998). GM-CSFadministration may be useful in the treatment of prostate cancer (Smallet al., 1999). GM-CSF monotherapy also has been reported to reducemelanoma metastases (Hoeller et al., 2001). GM-CSF also may be useful inthe treatment of melanoma when co-administered with anti-neoplasticagents (Vaughan et al., 2000). The immunomodulatory properties of GM-CSFalso suggest that PEGylated human GM-CSF cysteine analogs may haveutility as an adjuvant for vaccines against tumors and infectious agents(Gaudernack and Gjertsen, 1999, Warren and Weiner, 2000). For exampleadjuvant therapy using GM-CSF may be useful in treatment of malignantmelanoma (Spitler et al., 2000) following surgical resection. GM-CSFco-administration with a monoclonal antibody-based immunogen may beuseful in the treatment of metastatic coleorectal cancer (Rucker et al.1999). The immunomodulatory properties of GM-CSF indicate that PEGylatedhuman GM-CSF cysteine analogs may also provide useful therapy forenhancing immune function in immunocompromised patients such as thoseinfected with the HIV virus (Armitage, 1998). The immunomodulatoryproperties of GM-CSF indicate that PEGylated human GM-CSF cysteineanalogs may also provide useful therapy against bacterial and fungalinfections (Armitage, 1998; Jones, 1999).

GM-CSF also may be used to accelerate wound healing of surgicalincisions (Jyung et al., 1994) and also to accelerate healing ofrefractory wounds or ulcers such as those of patients with diabetesCanturk et al., 1999) and PEGylated human GM-CSF cysteine analogs mayalso be used in these indications.

In some diseases, antagonists of GM-CSF activity could providetherapeutic benefit. For example there is evidence that rheumatoidarthritis is promoted by GM-CSF activity (Campbell et al., 1997; Bischofet al., 2000; Campbell et al., 1998; Yang and Hamilton, 2001) and ananti-GM-CSF antibody has been shown to reduce the severity of arthritisobserved in a murine model (Cook et al., 2001). Therefore PEGylatedhuman GM-CSF cysteine analogs that contain one or more additionalmutations that result in GM-CSF antagonist activity could be used totreat arthritis. GM-CSF activity is similarly implicated in thedevelopment and progression of multiple sclerosis (McQualter et al.,2001) and PEGylated human GM-CSF cysteine analogs that contain one ormore additional mutations that result in GM-CSF antagonist activitycould be used to treat multiple sclerosis.

As discussed above, various embodiments of the invention relate tomethods of use of GM-CSF cysteine muteins. In particular, the presentinvention relates to the use of these muteins to protect an animal froma disease or condition that is amenable to treatment by the use ofwild-type GM-CSF or an antagonist thereof, or which might beparticularly amenable to treatment using the GM-CSF cysteine muteins ofthe present invention.

As used herein, the phrase “protected from a disease” refers to reducingthe symptoms of the disease, reducing the occurrence of the disease,and/or reducing the severity of the disease. Protecting an animal canrefer to the ability of a therapeutic composition of the presentinvention, when administered to an animal, to prevent a disease fromoccurring and/or to cure or to alleviate disease symptoms, signs orcauses. As such, to protect an animal from a disease includes bothpreventing disease occurrence (propylacetic treatment) and treating ananimal that has a disease or that is experiencing initial symptoms of adisease (therapeutic treatment). In particular, protecting an animalfrom a disease is accomplished by inducing a beneficial or protectivetherapeutic response in the animal by administration of a GM-CSFcysteine mutein of the present invention. The term, “disease” refers toany deviation from the normal health of a mammal and includes a statewhen disease symptoms are present, as well as conditions in which adeviation (e.g., infection, gene mutation, genetic defect, etc.) hasoccurred, but symptoms are not yet manifested.

Accordingly, a GM-CSF cysteine mutein of the present invention can beadministered to regulate the proliferation and differentiation ofhematopoietic progenitor cells into mature cells such as neutrophils,macrophages and eosinophils. A GM-CSF cysteine mutein can also beadministered to stimulate the functional properties of mature monocytes,neutrophils and eosinophils. A GM-CSF cysteine mutein, or antagonistthereof, can be administered to an animal to prevent or ameliorate anydisease or condition for which the use of the wild-type protein, orantagonist thereof, respectively, can be used. Such diseases andconditions are described in detail above. In one embodiment, a GM-CSFcysteine mutein of the present invention is used as an anti-neoplasticagent either alone or in combination with other anti-neoplastic agentssuch as cytotoxic drugs, chemotherapeutic agents, polyclonal antibodiesand monoclonal antibodies targeting tumor cells. In yet anotherembodiment, the GM-CSF cysteine mutein of the present invention is usedas an adjuvant for vaccines against tumors and infectious agents.

Cysteine muteins of the present invention are preferably administered ina composition. Compositions can include a cysteine mutein of theinvention and any other suitable pharmaceutically acceptable carrier, aswell as, in some aspects, additional components that may be useful inthe treatment of a give disease or condition. According to the presentinvention, a “pharmaceutically acceptable carrier” includespharmaceutically acceptable excipients and/or pharmaceuticallyacceptable delivery vehicles, which are suitable for use inadministration of the composition to a suitable in vitro, ex vivo or invivo site. A suitable in vitro, in vivo or ex vivo site is preferablyany site where the cysteine mutein will provide a detectable effect ascompared to in the absence of the mutein, and includes a disease site ora site of cell types to be contacted with the mutein. Preferredpharmaceutically acceptable carriers are capable of maintaining themutein of the present invention in a form that, upon arrival of themutein at the cell target in a culture or in patient, the mutein iscapable of interacting with its target (e.g., hematopoeitic cell forGM-CSF).

Suitable excipients of the present invention include excipients orformularies that transport or help transport, but do not specificallytarget a composition to a cell or area (also referred to herein asnon-targeting carriers). Examples of pharmaceutically acceptableexcipients include, but are not limited to water, phosphate bufferedsaline, Ringer's solution, dextrose solution, serum-containingsolutions, Hank's solution, other aqueous physiologically balancedsolutions, oils, esters and glycols. Aqueous carriers can containsuitable auxiliary substances required to approximate the physiologicalconditions of the recipient, for example, by enhancing chemicalstability and isotonicity. Compositions of the present invention can besterilized by conventional methods and/or lyophilized.

One type of pharmaceutically acceptable carrier includes a controlledrelease formulation that is capable of slowly releasing a composition ofthe present invention into a patient or culture. As used herein, acontrolled release formulation comprises a cysteine mutein of thepresent invention in a controlled release vehicle. Suitable controlledrelease vehicles include, but are not limited to, biocompatiblepolymers, other polymeric matrices, capsules, microcapsules,microparticles, bolus preparations, osmotic pumps, diffusion devices,liposomes, lipospheres, and transdermal delivery systems. Other carriersof the present invention include liquids that, upon administration to apatient, form a solid or a gel in situ. Preferred carriers are alsobiodegradable (i.e., bioerodible). In the event that a cysteine muteinof the invention is administered as a recombinant nucleic acid moleculeencoding the cysteine mutein (e.g., gene therapy or geneticimmunization), suitable carriers include, but are not limited toliposomes, viral vectors or other carriers, including ribozymes, goldparticles, poly-L-lysine/DNA-molecular conjugates, and artificialchromosomes. Natural lipid-containing carriers include cells andcellular membranes. Artificial lipid-containing carriers includeliposomes and micelles.

A carrier of the present invention can be modified to target to aparticular site in a patient, thereby targeting and making use of acompound of the present invention at that site. A pharmaceuticallyacceptable carrier which is capable of targeting can also be referred toherein as a “delivery vehicle” or “targeting carrier”. Suitablemodifications include manipulating the chemical formula of the lipidportion of the delivery vehicle and/or introducing into the vehicle atargeting agent capable of specifically targeting a delivery vehicle toa preferred site or target site, for example, a preferred cell type. A“target site” refers to a site in a patient to which one desires todeliver a composition. Suitable targeting compounds include ligandscapable of selectively (i.e., specifically) binding another molecule ata particular site. Examples of such ligands include antibodies,antigens, receptors and receptor ligands. Manipulating the chemicalformula of the lipid portion of the delivery vehicle can modulate theextracellular or intracellular targeting of the delivery vehicle. Forexample, a chemical can be added to the lipid formula of a liposome thatalters the charge of the lipid bilayer of the liposome so that theliposome fuses with particular cells having particular chargecharacteristics.

One delivery vehicle of the present invention is a liposome. A liposomeis capable of remaining stable in an animal for a sufficient amount oftime to deliver a nucleic acid molecule or protein described in thepresent invention to a preferred site in the animal. A liposome,according to the present invention, comprises a lipid composition thatis capable of delivering a nucleic acid molecule or protein to aparticular, or selected, site in a patient. A liposome according to thepresent invention comprises a lipid composition that is capable offusing with the plasma membrane of the targeted cell to deliver anucleic acid molecule or protein into a cell. Suitable liposomes for usewith the present invention include any liposome. Preferred liposomes ofthe present invention include those liposomes commonly used in, forexample, gene delivery methods known to those of skill in the art. Morepreferred liposomes comprise liposomes having a polycationic lipidcomposition and/or liposomes having a cholesterol backbone conjugated topolyethylene glycol. Complexing a liposome with a nucleic acid moleculeor protein of the present invention can be achieved using methodsstandard in the art.

Another type of delivery vehicle, when the cysteine mutein isadministered as a nucleic acid encoding the mutein, comprises a viralvector. A viral vector includes an isolated nucleic acid molecule, inwhich the nucleic acid molecules are packaged in a viral coat thatallows entrance of DNA into a cell. A number of viral vectors can beused, including, but not limited to, those based on alphaviruses,poxviruses, adenoviruses, herpesviruses, lentiviruses, adeno-associatedviruses and retroviruses.

According to the present invention, an effective administration protocol(i.e., administering a therapeutic composition in an effective manner)comprises suitable dose parameters and modes of administration thatresult in the desired effect in the patient (e.g., stimulation ofproliferation and/or differentiation of neutrophils), preferably so thatthe patient is protected from the disease (e.g., by disease preventionor by alleviating one or more symptoms of ongoing disease). Effectivedose parameters can be determined using methods standard in the art fora particular disease. Such methods include, for example, determinationof survival rates, side effects (i.e., toxicity) and progression orregression of disease.

In accordance with the present invention, a suitable single dose size isa dose that results in the desired therapeutic effect in the patient,depending o the cysteine mutein that is administered, or in theamelioration of at least one symptom of a condition in the patient, whenadministered one or more times over a suitable time period. Doses canvary depending upon the disease being treated. One of skill in the artcan readily determine appropriate single dose sizes for a given patientbased on the size of a patient and the route of administration.

In one aspect of the invention, a suitable single dose of a therapeuticcomposition of the present invention is an amount that, whenadministered by any route of administration, provides a therapeuticeffect in the patient as described above, as compared to a patient whichhas not been administered with the therapeutic composition of thepresent invention (i.e., a control patient), as compared to the patientprior to administration of the composition, or as compared to a standardestablished for the particular disease, patient type and composition.

In one aspect of the invention an appropriate single dose of a cysteinemutein of the present invention is at least about 0.01 μg per kg of theanimal to which the mutein is administered, and in other aspects, atleast about 0.1 μg/kg, at least about 0.2 μg/kg, at least about 0.5μg/kg, at least about 1 μg/kg, at least about 5 μg/kg, at least about 10μg/kg, at least about 25 μg/kg, at least about 50 μg/kg, at least about75 μg/kg, at least about 100 μg/kg, at least about 200 μg/kg, at leastabout 300 μg/kg, at least about 400 μg/kg, at least about 500 μg/kg, atleast about 750 μg/kg, at least about 1 mg/kg, or at least about 5mg/kg. In one embodiment, a preferred single dose of a GM-CSF cysteinemutein of the present invention is at least about 0.1 μg/kg, and morepreferably, from about 25 to about 300 μg/kg.

As discussed above, a therapeutic composition of the present inventionis administered to a patient in a manner effective to deliver thecomposition to a cell, a tissue, and/or systemically to the patient,whereby the desired result is achieved as a result of the administrationof the composition. Suitable administration protocols include any invivo or ex vivo administration protocol. The preferred routes ofadministration will be apparent to those of skill in the art, dependingon the type of condition to be prevented or treated; whether thecomposition is nucleic acid based or protein based; and/or the targetcell/tissue. For proteins or nucleic acid molecules, preferred methodsof in vivo administration include, but are not limited to, intravenousadministration, intraperitoneal administration, intramuscularadministration, intranodal administration, intracoronary administration,intraarterial administration (e.g., into a carotid artery), subcutaneousadministration, transdermal delivery, intratracheal administration,subcutaneous administration, intraarticular administration,intraventricular administration, inhalation (e.g., aerosol),intracranial, intraspinal, intraocular, intranasal, oral, bronchial,rectal, topical, vaginal, urethral, pulmonary administration,impregnation of a catheter, and direct injection into a tissue. Routesuseful for deliver to mucosal tissues include, bronchial, intradermal,intramuscular, intranasal, other inhalatory, rectal, subcutaneous,topical, transdermal, vaginal and urethral routes. Combinations ofroutes of delivery can be used and in some instances, may enhance thetherapeutic effects of the composition. Particularly preferred routes ofdelivery include subcutaneous and intravenous delivery.

Ex vivo administration refers to performing part of the regulatory stepoutside of the patient, such as administering a composition of thepresent invention to a population of cells removed from a patient underconditions such that the composition contacts and/or enters the cell,and returning the cells to the patient. Ex vivo methods are particularlysuitable when the target cell type can easily be removed from andreturned to the patient.

Many of the above-described routes of administration, includingintravenous, intraperitoneal, intradermal, and intramuscularadministrations can be performed using methods standard in the art.Aerosol (inhalation) delivery can also be performed using methodsstandard in the art (see, for example, Stribling et al., Proc. Natl.Acad. Sci. USA 189:11277-11281, 1992, which is incorporated herein byreference in its entirety). Oral delivery can be performed by complexinga therapeutic composition of the present invention to a carrier capableof withstanding degradation by digestive enzymes in the gut of ananimal. Examples of such carriers, include plastic capsules or tablets,such as those known in the art.

One method of local administration is by direct injection. Directinjection techniques are particularly useful for administering acomposition to a cell or tissue that is accessible by surgery, andparticularly, on or near the surface of the body. Administration of acomposition locally within the area of a target cell refers to injectingthe composition centimeters and preferably, millimeters from the targetcell or tissue.

Various methods of administration and delivery vehicles disclosed hereinhave been shown to be effective for delivery of a nucleic acid moleculeto a target cell, whereby the nucleic acid molecule transfected the celland was expressed. In many studies, successful delivery and expressionof a heterologous gene was achieved in preferred cell types and/or usingpreferred delivery vehicles and routes of administration of the presentinvention. All of the publications discussed below and elsewhere hereinwith regard to gene delivery and delivery vehicles are incorporatedherein by reference in their entirety.

For example, using liposome delivery, U.S. Pat. No. 5,705,151, issuedJan. 6, 1998, to Dow et al. demonstrated the successful in vivointravenous delivery of a nucleic acid molecule encoding a superantigenand a nucleic acid molecule encoding a cytokine in a cationic liposomedelivery vehicle, whereby the encoded proteins were expressed in tissuesof the animal, and particularly in pulmonary tissues. In addition, Liuet al., Nature Biotechnology 15:167, 1997, demonstrated that intravenousdelivery of cholesterol-containing cationic liposomes containing genespreferentially targets pulmonary tissues and effectively mediatestransfer and expression of the genes in vivo. Several publications byDzau and collaborators demonstrate the successful in vivo delivery andexpression of a gene into cells of the heart, including cardiac myocytesand fibroblasts and vascular smooth muscle cells using both naked DNAand Hemagglutinating virus of Japan-liposome delivery, administered byboth incubation within the pericardium and infusion into a coronaryartery (intracoronary delivery) (See, for example, Aoki et al., 1997, J.Mol. Cell, Cardiol. 29:949-959; Kaneda et al., 1997, Ann N.Y. Acad. Sci.811:299-308; and von der Leyen et al., 1995, Proc Natl Acad Sci USA92:1137-1141).

Delivery of numerous nucleic acid sequences has been accomplished byadministration of viral vectors encoding the nucleic acid sequences.Using such vectors, successful delivery and expression has been achievedusing ex vivo delivery (See, of many examples, retroviral vector; Blaeseet al., 1995, Science 270:475-480; Bordignon et al., 1995, Science270:470-475), nasal administration (CFTR-adenovirus-associated vector),intracoronary administration (adenoviral vector and Hemagglutinatingvirus of Japan, see above), intravenous administration (adeno-associatedviral vector; Koeberl et al., 1997, Proc Natl Acad Sci USA94:1426-1431). A publication by Maurice et al. (1999, J. Clin. Invest.104:21-29) demonstrated that an adenoviral vector encoding aβ2-adrenergic receptor, administered by intracoronary delivery, resultedin diffuse multichamber myocardial expression of the gene in vivo, andsubsequent significant increases in hemodynamic function and otherimproved physiological parameters. Levine et al. describe in vitro, exvivo and in vivo delivery and expression of a gene to human adipocytesand rabbit adipocytes using an adenoviral vector and direct injection ofthe constructs into adipose tissue (Levine et al., 1998, J. Nutr. Sci.Vitaminol. 44:569-572).

In the area of neuronal gene delivery, multiple successful in vivo genetransfers have been reported. Millecamps et al. reported the targetingof adenoviral vectors to neurons using neuron restrictive enhancerelements placed upstream of the promoter for the transgene(phosphoglycerate promoter). Such vectors were administered to mice andrats intramuscularly and intracerebrally, respectively, resulting insuccessful neuronal-specific transfection and expression of thetransgene in vivo (Millecamps et al., 1999, Nat. Biotechnol.17:865-869). As discussed above, Bennett et al. reported the use ofadeno-associated viral vector to deliver and express a gene bysubretinal injection in the neural retina in vivo for greater than 1year (Bennett, 1999, ibid.).

Gene delivery to synovial lining cells and articular joints has hadsimilar successes. Oligino and colleagues report the use of a herpessimplex viral vector which is deficient for the immediate early genes,ICP4, 22 and 27, to deliver and express two different receptors insynovial lining cells in vivo (Oligino et al., 1999, Gene Ther.6:1713-1720). The herpes vectors were administered by intraarticularinjection. Kuboki et al. used adenoviral vector-mediated gene transferand intraarticular injection to successfully and specifically express agene in the temporomandibular joints of guinea pigs in vivo (Kuboki etal., 1999, Arch. Oral. Biol. 44:701-709). Apparailly and colleaguessystemically administered adenoviral vectors encoding IL-10 to mice anddemonstrated successful expression of the gene product and profoundtherapeutic effects in the treatment of experimentally induced arthritis(Apparailly et al., 1998, J. Immunol. 160:5213-5220). In another study,murine leukemia virus-based retroviral vector was used to deliver (byintraarticular injection) and express a human growth hormone gene bothex vivo and in vivo (Ghivizzani et al., 1997, Gene Ther. 4:977-982).This study showed that expression by in vivo gene transfer was at leastequivalent to that of the ex vivo gene transfer. As discussed above,Sawchuk et al. has reported successful in vivo adenoviral vectordelivery of a gene by intraarticular injection, and prolonged expressionof the gene in the synovium by pretreatment of the joint with anti-Tcell receptor monoclonal antibody (Sawchuk et al., 1996, ibid. Finally,it is noted that ex vivo gene transfer of human interleukin-1 receptorantagonist using a retrovirus has produced high level intraarticularexpression and therapeutic efficacy in treatment of arthritis, and isnow entering FDA approved human gene therapy trials (Evans and Robbins,1996, Curr. Opin. Rheumatol. 8:230-234). Therefore, the state of the artin gene therapy has led the FDA to consider human gene therapy anappropriate strategy for the treatment of at least arthritis. Takentogether, all of the above studies in gene therapy indicate thatdelivery and expression of a recombinant nucleic acid molecule accordingto the present invention is feasible.

Another method of delivery of recombinant molecules is in anon-targeting carrier (e.g., as “naked” DNA molecules, such as istaught, for example in Wolff et al., 1990, Science 247, 1465-1468). Suchrecombinant nucleic acid molecules are typically injected by direct orintramuscular administration. Recombinant nucleic acid molecules to beadministered by naked DNA administration include an isolated nucleicacid molecule of the present invention, and preferably includes arecombinant molecule of the present invention that preferably isreplication, or otherwise amplification, competent. A naked nucleic acidreagent of the present invention can comprise one or more nucleic acidmolecules of the present invention including a dicistronic recombinantmolecule. Naked nucleic acid delivery can include intramuscular,subcutaneous, intradermal, transdermal, intranasal and oral routes ofadministration, with direct injection into the target tissue being mostpreferred. A preferred single dose of a naked nucleic acid vaccineranges from about 1 nanogram (ng) to about 100 μg, depending on theroute of administration and/or method of delivery, as can be determinedby those skilled in the art. Suitable delivery methods include, forexample, by injection, as drops, aerosolized and/or topically. In oneembodiment, pure DNA constructs cover the surface of gold particles (1to 3 μm in diameter) and are propelled into skin cells or muscle with a“gene gun.”

In the method of the present invention, compositions can be administeredto any animal and preferably, to any member of the Vertebrate class,Mammalia, including, without limitation, primates, rodents, livestockand domestic pets. Livestock include mammals to be consumed or thatproduce useful products (e.g., sheep for wool production). Preferredmammals to protect include humans, dogs, cats, mice, rats, sheep,cattle, horses and pigs, with humans being particularly preferred.

The following examples are provided to demonstrate how the “rules”described herein can be used to create cysteine-added variants of GH,erythropoietin, alpha interferon, beta interferon, G-CSF, GM-CSF andother members of the GH supergene family. The examples also demonstratethe therapeutic uses of cysteine variants of the present invention. Theexamples are not intended to be limiting, but only exemplary of specificembodiments of the invention.

EXAMPLE 1 Cysteine-Added Variants of GH

This example discloses certain amino acids in GH that are non-essentialfor biological activity and which, when mutated to cysteine residues,will not alter the normal disulfide binding pattern and overallconformation of the molecule. These amino acids are located at theN-terminal end of the A-B loop (amino acids 34-52 of the mature proteinsequence; SEQ ID NO: 1; Martial et al 1979; Goeddel et al 1979), the B-Cloop (amino acids 97-105 in the mature protein sequence), and the C-Dloop (amino acids 130-153 in the mature protein sequence). Alsoidentified as preferred sites for introducing cysteine residues are thefirst three or last three amino acids in the A, B, C and D helices andthe amino acids proximal to helix A (amino acids 1-5) and distal tohelix D (amino acids 184-191). Helix A encompasses amino acids 6-33,helix B encompasses amino acids 75-96, helix C encompasses amino acids106-129 and helix D encompasses amino acids 154-183. The entire A-B loopencompasses amino acids 34-74.

DNA sequences encoding wild type GH can be amplified using thepolymerase chain reaction technique from commercially availablesingle-stranded cDNA prepared from human pituitaries (ClonTech, SanDiego, Calif.) or assembled using overlapping oligonucleotides. Specificmutations can be introduced into the GH sequence using a variety ofprocedures such as phage techniques (Kunkel et al 1987), PCR mutagenesistechniques (Innis et al 1990; White 1993) mutagenesis kits such as thosesold by Stratagene (“Quick-Change Mutagenesis” kit, San Diego, Calif.)or Promega (Gene Editor Kit, Madison Wis.).

Cysteine substitutions can be introduced into any of the amino acidscomprising the B-C loop, C-D loop and N-terminal end of the A-B loop orinto the first three amino acids of the alpha helical regions thatadjoin these regions, or in the region proximal to helix A or distal tohelix D. Preferred sites for introduction of cysteine residues are: F1,T3, P5, E33, A34, K38, E39, Q40, S43, Q46, N47, P48, Q49, T50, S51, S55,T60, A98, N99, S100, G104, A105, S106, E129, D130, G131, S132, P133,T135, G136, Q137, K140, Q141, T142, S144, K145, D147, T148, N149, S150,H151, N152, D153, S184, E186, G187, S188, and G190. Cysteine residuesalso can be introduced at the beginning of the mature protein, i.e.,proximal to the F1 amino acid, or following the last amino acid in themature protein, i.e., following F191. If desirable, two or more suchmutations can be readily combined in the same protein either by in vitroDNA recombination of cloned mutant genes and/or sequential constructionof individual desired mutations.

1. Cloning the Gene for Human Growth Hormone (GH)

The human GH gene was amplified from human pituitary single-strandedcDNA (commercially available from CLONTECH, Inc., Palo Alto, Calif.)using the polymerase chain reaction (PCR) technique and primers BB1 andBB2. The sequence of BB1 is5′-GGGGGTCGACCATATGTTCCCAACCATTCCCTTATCCAG-3′ (SEQ ID NO: 24). Thesequence of BB2 is 5′-GGGGGATCCTCACTAGAAGCCACAGCTGCCCTC-3′ (SEQ ID NO:25). Primer BB1 was designed to encode an initiator methionine precedingthe first amino acid of mature GH, phenylalanine, and SalI and NdeIsites for cloning purposes. The reverse primer, BB2, contains a BamHIsite for cloning purposes. The PCR 100 microliter reactions contained 20pmoles of each oligonucleotide primer, 1×PCR buffer (Perkin-Elmer buffercontaining MgCl₂), 200 micromolar concentration of each of the fournucleotides dA, dC, dG and dT, 2 ng of single-stranded cDNA, 2.5 unitsof Taq polymerase (Perkin-Elmer) and 2.5 units of Pfu polymerase(Stratagene, Inc). The PCR reaction conditions were 96° C. for 3minutes, 35 cycles of (95° C., 1 minute; 63° C. for 30 seconds; 72° C.for 1 minute), followed by 10 minutes at 72° C. The thermocycleremployed was the Amplitron II Thermal Cycler (Thermolyne). Theapproximate 600 bp PCR product was digested with SalI and BamHI, gelpurified and cloned into similarly digested plasmid pUC19 (commerciallyavailable from New England BioLabs, Beverly, Mass.). The ligationmixture was transformed into E. coli strain DH5alpha and transformantsselected on LB plates containing ampicillin. Several colonies were grownovernight in LB media and plasmid DNA isolated using miniplasmid DNAisolation kits purchased from Qiagen, Inc (Valencia, Calif.). Clone LB6was determined to have the correct DNA sequence.

For expression in E. coli, clone LB6 was digested with NdeI and EcoRI,the approximate 600 bp fragment gel-purified, and cloned into plasmidpCYB1 (commercially available from New England BioLabs, Beverly, Mass.)that had been digested with the same enzymes and phosphatased. Theligation mixture was transformed into E. coli DH5alpha and transformantsselected on LB ampicillin plates. Plasmid DNA was isolated from severaltransformants and screened by digestion with NdeI and EcoRI. A correctclone was identified and named pCYB1: wtGH (pBBT120). This plasmid wastransformed into E. coli strains JM109 or W3110 (available from NewEngland BioLabs and the American Type Culture Collection).

2. Construction of STII-GH

Wild type GH clone LB6 (pUC19: wild type GH) was used as the template toconstruct a GH clone containing the E. coli STII signal sequence (Pickenet al. 1983). Because of its length, the STII sequence was added in twosequential PCR reactions. The first reaction used forward primer BB12and reverse primer BB10. BB10 has the sequence:5′CGCGGATCCGATTAGAATCCACAGCTCCCCTC 3′ (SEQ ID NO: 28).

BB12 has the sequence: (SEQ ID NO: 30)5′ATCTATGTTCGTTTTCTCTATCGCTACCAACGCTTACGCATTCCCAAC CATTCCCTTATCCAG-3′.

The PCR reactions were as described for amplifying wild type GH exceptthat approximately 4 ng of plasmid LB6 was used as the template ratherthan single-stranded cDNA and the PCR conditions were 96° C. for 3minutes, 30 cycles of (95° C. for 1 minute; 63° C. for 30 seconds; 72°C. for 1 minute) followed by 72° C. for 10 minutes. The approximate 630bp PCR product was gel-purified using the Qiaex II Gel Extraction Kit(Qiagen, Inc), diluted 50-fold in water and 2 microliters used astemplate for the second PCR reaction. The second PCR reaction usedreverse primer BB10 and forward primer BB11. BB11 has the sequence: (SEQID NO: 29) 5′CCCCCTCTAGACATATGAAGAAGAACATCGCATTCCTGCTGGCATCTATGTTCGTTTTCTCTATCG-3′.

Primer BB11 contains XbaI and NdeI sites for cloning purposes. PCRconditions were as described for the first reaction. The approximate 660bp PCR product was digested with XbaI and BamHI, gel-purified and clonedinto similarly cut plasmid pCDNA3.1(+) (Invitrogen, Inc. Carlsbad,Calif.). Clone pCDNA3.1(+)::stII-GH(5C) or “5C” was determined to havethe correct DNA sequence.

Clone “5C” was cleaved with NdeI and BamHI and cloned into similarly cutpBBT108 (a derivative of pUC19 which lacks a Pst I site, this plasmid isdescribed below). A clone with the correct insert was identifiedfollowing digestion with these enzymes. This clone, designated pBBT111,was digested with NdeI and SalI, the 660 bp fragment containing thestII-GH fusion gene, was gel-purified and cloned into the plasmidexpression vector pCYB1 (New England BioLabs) that had been digestedwith the same enzymes and phosphatased. A recombinant plasmid containingthe stII-GH insertion was identified by restriction endonucleasedigestions. One such isolate was chosen for further studies and wasdesignated pBBT114. This plasmid was transformed into E. coli strainsJM109 or W3110 (available from New England BioLabs and the American TypeCulture Collection).

3. Construction of ompA-GH

Wild type GH clone LB6 (pUC19: wild type GH) was used as the template toconstruct a GH clone containing the E. coli ompA signal sequence (Movvaet al 1980). Because of its length, the ompA sequence was added in twosequential PCR reactions. The first reaction used forward primer BB7:(SEQ ID NO: 31) 5′GCAGTGGCACTGGCTGGTTTCGCTACCGTAGCGCAGGCCTTCCCAACCATTCCCTTATCCAG 3′,

and reverse primer BB10: (SEQ ID NO: 28)5′CGCGGATCCGATTAGAATCCACAGCTCCCCTC 3′.

The PCR reactions were as described for amplifying wild type GH exceptthat approximately 4 ng of plasmid LB6 was used as the template ratherthan single-stranded cDNA and the PCR conditions were 96° C. for 3minutes, 30 cycles of (95° C. for 1 minute; 63° C. for 30 seconds; 72°C. for 1 minute) followed by 72° C. for 10 minutes. The approximate 630bp PCR product was gel-purified using the Qiaex II Gel Extraction Kit(Qiagen, Inc), diluted 50-fold in water and 2 microliters used astemplate for the second PCR reaction. The second PCR reaction usedreverse primer BB10 and forward PrimerBB6: (SEQ ID NO: 32)5′CCCCGTCGACACATATGAAGAAGACAGCTATCGCGATTGCAGTGGCAC TGGCTGGTTTC 3′

PCR conditions were as described for the first reaction. The approximate660 bp PCR product was gel-purified, digested with Sal I and Bam H1 andcloned into pUC19 (New England BioLabs) which was cut with Sal I and BamH1 or pCDNA3.1(+) (Invitrogen) which had been cut by Xho I and Bam H1(Sal I and Xho I produce compatible single-stranded overhangs). Whenseveral clones were sequenced, it was discovered that all pUC19 clones(8/8) contained errors in the region of the ompA sequence. Only onepCDNA3.1(+) clone was sequenced and it contained a sequence ambiguity inthe ompA region. In order to generate a correct ompA-GH fusion genesegments of two sequenced clones which contained different errorsseparated by a convenient restriction site were recombined and clonedinto the pUC19-derivative that lacks the Pst I site (see pBBT108described below). The resulting plasmid, termed pBBT112, carries theompA-GH fusion gene cloned as an Nde I-Bam H1 fragment into these samesites in pBBT108. This plasmid is designated pBBT112 and is used inPCR-based, site-specific mutagenesis of GH as described below.

4. Construction of Pst-pUC19

To facilitate mutagenesis of the cloned GH gene for construction ofselected cysteine substitution and insertion mutations a derivative ofthe plasmid pUC19 (New England BioLabs) lacking a Pst I site wasconstructed as follows. pUC19 plasmid DNA was digested with Pst I andsubsequently treated at 75 deg. C. with PFU DNA Polymerase (Stratagene)using the vendor-supplied reaction buffer supplemented with 200 uMdNTPs. Under these conditions the polymerase will digest the 3′single-stranded overhang created by Pst I digestion but will not digestinto the double-stranded region. The net result will be the deletion ofthe 4 single-stranded bases which comprise the middle four bases of thePst I recognition site. The resulting molecule has double-stranded,i.e., “blunt”, ends. Following these enzymatic reactions, the linearmonomer was gel-purified using the Qiaex II Gel Extraction Kit (Qiagen,Inc). This purified DNA was treated with T4 DNA Ligase (New EnglandBioLabs) according to the vendor protocols, digested with Pst I, andused to transform E coli DH5alpha. Transformants were picked andanalyzed by restriction digestion with Pst I and Bam H1. One of thetransformants which was not cleaved by Pst I but was cleaved at thenearby Bam H1 site was picked and designated pBBT108.

5. Construction of GH Muteins

GH muteins were generally constructed using site-directed PCR-basedmutagenesis as described in PCR Protocols: Current Methods andApplications edited by B. A. White, 1993 Humana Press, Inc., Totowa,N.J. and PCR Protocols: A Guide to Methods and Applications edited byInnis, M. A. et al 1990 Academic Press Inc San Diego, Calif. TypicallyPCR primer oligonucleotides are designed to incorporate nucleotidechanges to the coding sequence of GH that result in substitution of acysteine residue for an amino acid at a specific position within theprotein. Such mutagenic oligonucleotide primers can also be designed toincorporate an additional cysteine residue at the carboxy terminus oramino terminus of the coding sequence of GH. In this latter case one ormore additional amino acid residues could also be incorporated at theamino terminal and/or carboxy terminal to the added cysteine residue ifthat were desirable. Moreover, oligonucleotides can be designed toincorporate cysteine residues as insertion mutations at specificpositions within the GH coding sequence if that were desirable. Again,one or more additional amino acids could be inserted along with thecysteine residue and these amino acids could be positioned at the aminoterminal and/or carboxy terminal to the cysteine residue.

The cysteine substitution mutation T135C was constructed as follows. Themutagenic reverse oligonucleotide BB28: (SEQ ID NO: 33)5′CTGCTTGAAGATCTGCCCACACCGGGGGCTGCCATC 3′was designed to change the codon ACT for threonine at amino acid residue135 to a TGT codon encoding cysteine and to span the nearby Bgl II site.This oligonucleotide was used in PCR along with the forwardoligonucleotide BB34 5′GTAGCGCAGGCCTTCCCAACCATT3′ (SEQ ID NO: 34) whichanneals to the junction region of the ompA-GH fusion gene and is notmutagenic. The PCR was performed in a 50 ul reaction in 1×PCR buffer(Perkin-Elmer buffer containing 1.5 mM MgCl₂), 200 micromolarconcentration of each of the four nucleotides dA, dC, dG and dT, witheach oligonucleotide primer present at 0.5 μM, 5 pg of pBBT112(described above) as template and 1.25 units of Amplitac DNA Polymerase(Perkin-Elmer) and 0.125 units of PFU DNA Polymerase (Stratagene).Reactions were performed in a Robocycler Gradient 96 thermal cycler(Stratagene). The program used entailed: 95 deg C. for 3 minutesfollowed by 25 cycles of 95 deg C. for 60 seconds, 45 deg C. or 50 degC. or 55 deg C. for 75 seconds, 72 deg C. for 60 seconds followed by ahold at 6 deg C. The PCR reactions were analyzed by agarose gelelectrophoresis to identify annealing temperatures that gave significantproduct of the expected size; ˜430 bp. The 45-deg C. reaction was“cleaned up” using the QIAquick PCR Purification Kit (Qiagen), digestedwith Bgl II and Pst I. The resulting 278 bp Bgl II-Pst I fragment, whichincludes the putative T135C mutation, was gel-purified and ligated intopBBT111 the pUC19 derivative carrying the stII-GH fusion gene (describedabove) which had been digested with Bgl II and Pst I and gel-purified.Transformants from this ligation were initially screened by digestionwith Bgl II and Pst I and subsequently one clone was sequenced toconfirm the presence of the T135C mutation and the absence of anyadditional mutations that could potentially be introduced by the PCRreaction or by the synthetic oligonucleotides. The sequenced clone wasfound to have the correct sequence.

The substitution mutation S132C was constructed using the protocoldescribed above for T135C with the following differences: mutagenicreverse oligonucleotide BB29 5′CTGCTTGAAGATCTGCCCAGTCCGGGGGCAGCCATCTTC3′(SEQ ID NO: 35) was used instead of BB28 and the PCR reaction withannealing temperature of 50 deg C. was used for cloning. One of twoclones sequenced was found to have the correct sequence.

The substitution mutation T148C was constructed using an analogousprotocol but employing a different cloning strategy. The mutagenicforward oligonucleotide BB305′GGGCAGATCTTCAAGCAGACCTACAGCAAGTTCGACTGCAACTCACACAAC3′ (SEQ ID NO: 36)was used in PCR with the non-mutagenic reverse primer BB335′CGCGGTACCCGGGATCCGATTAGAATCCACAGCT3′ (SEQ ID NO: 37) which anneals tothe most 3′ end of the GH coding sequence and spans the Bam H1 siteimmediately downstream. PCR was performed as described above with theexception that the annealing temperatures used were 46, 51 and 56 deg C.Following PCR and gel analysis as described above the 46 and 51 deg C.reactions were pooled for cloning. These were digested with Bam H1 andBgl II, gel-purified and cloned into pBBT111 which had been digestedwith Bam H1 and Bgl II, treated with Calf intestinal AlkalinePhosphatase (Promega) according to the vendor protocols, andgel-purified. Transformants from this ligation were analyzed bydigestion with Bam H1 and Bgl II to identify clones in which the 188 bpBam H1-Bgl II mutagenic PCR fragment was cloned in the properorientation. Because Bam H1 and Bgl II generate compatible ends, thiscloning step is not orientation specific. Five of six clones tested wereshown to be correctly oriented. One of these was sequenced and was shownto contain the desired T148C mutation. The sequence of the remainder ofthe 188 bp Bam H1-Bgl II mutagenic PCR fragment in this clone wasconfirmed as correct.

The construction of the substitution mutation S144C was identical to theconstruction of T148C with the following exceptions. Mutagenic forwardoligonucleotide BB31 5′GGGCAGATCTTCAAGCAGACCTACTGCAAGTTCGAC3′ (SEQ IDNO: 38) was used instead of BB30. Two of six clones tested were shown tobe correctly oriented. One of these was sequenced and was shown tocontain the desired S144C mutation. The sequence of the remainder of the188 bp Bam H1-Bgl II mutagenic PCR fragment in this clone was confirmedas correct.

A mutation was also constructed that added a cysteine residue to thenatural carboxy terminus of GH. The construction of this mutation,termed stp192C, was similar to that of T148C, but employed differentoligonucleotide primers. The reverse mutagenic oligonucleotide: (SEQ IDNO: 39) BB32 5′CGCGGTACCGGATCCTTAGCAGAAGCCACAGCTGCCCTCCAC 3′which inserts a TGC codon for cysteine between the codon for the carboxyterminal phe residue of GH and the TAA translational stop codon andspans the nearby Bam H1 site was used along with BB345′GTAGCGCAGGCCTTCCCAACCATT3′ (SEQ ID NO: 40) which is described above.Following PCR and gel analysis as described above, the 46 deg C.reaction was used for cloning. Three of six clones tested were shown tobe correctly oriented. One of these was sequenced and was shown tocontain the desired stp192C mutation. The sequence of the remainder ofthe 188 bp Bam H1-Bgl II mutagenic PCR fragment in this clone wasconfirmed as correct.

Analogous PCR mutagenesis procedures can be used to generate othercysteine mutations. The choice of sequences for mutagenicoligonucleotides will be dictated by the position where the desiredcysteine residue is to be placed and the propinquity of usefulrestriction endonuclease sites. Generally, it is desirable to place themutation, i.e., the mismatched segment near the middle of theoligonucleotide to enhance the annealing of the oligonucleotide to thetemplate. Appropriate annealing temperatures for any oligonucleotide canbe determined empirically. It is also desirable for the mutagenicoligonucleotide to span a unique restriction site so that the PCRproduct can be cleaved to generate a fragment that can be readily clonedinto a suitable vector, e.g., one that can be used to express the muteinor provides convenient restriction sites for excising the mutated geneand readily cloning it into such an expression vector. Sometimesmutation sites and restriction sites are separated by distances that aregreater than that which is desirable for synthesis of syntheticoligonucleotides: it is generally desirable to keep sucholigonucleotides under 80 bases in length and lengths of 30-40 bases aremore preferable.

In instances where this is not possible, genes targeted for mutagenesiscould be re-engineered or re-synthesized to incorporate restrictionsites at appropriate positions. Alternatively, variations of PCRmutagenesis protocols employed above, such as the so-called “MegaprimerMethod” (Barik, S., pp. 277-286 in Methods in Molecular Biology, Vol.15: PCR Protocols: Current Methods and Applications edited by B. A.White, 1993, Humana Press, Inc., Totowa, N.J.) or “Gene Splicing byOverlap Extension” (Horton, R. M., pp. 251-261, in Methods in MolecularBiology, Vol. 15: PCR Protocols: Current Methods and Applications,edited by B. A. White, 1993, Humana Press, Inc., Totowa, N.J.) can alsobe employed to construct such mutations.

6. Expression of GH in pCYB1

To express GH in E. coli, pBBT120 (GH gene with no leader sequencecloned into the tac expression vector pCYB1) and pBBT114 (GH gene withstII leader sequence cloned into the tac expression vector pCYB1) weretransformed into E. coli strains JM109 and W3110. The parental vectorpCYB1 was also transformed into JM109 and W3110.

These strains were given the following designations: BOB119: JM109(pCYB1) BOB130: W3110 (pCYB1) BOB129: JM109 (pBBT120) BOB133: W3110(pBBT120) BOB121: JM109 (pBBT114) BOB132: W3110 (pBBT114)

For expression, strains were grown overnight at 37° C. in Luria Broth(LB) (Sambrook, et al 1989) containing 100 μg/ml ampicillin. Thesesaturated overnight cultures were diluted to ˜0.03 OD at A₆₀₀ in LBcontaining 100 μg/ml ampicillin and incubated at 37° C. in shake flasksin rotary shaker, typically at 250-300 rpm. ODs were monitored and IPTGwas added to a final concentration of 0.5 mM when culture ODs reached˜0.25-0.5, typically between 0.3 and 0.4. Cultures were sampledtypically at 1, 3, 5 and ˜16 h post-induction. The “˜16 h” time pointsrepresented overnight incubation of the cultures and exact times variedfrom ˜15-20 h. Samples of induced and uninduced cultures were pelletedby centrifugation, resuspended in 1× sample buffer (50 mM Tris-HCl (pH6.8), 2% sodium lauryl sulfate, 10% glycerol, 0.1% bromphenol blue) withthe addition of 1% β-mercaptoethanol when desirable. Samples were boiledfor ˜10 minutes or heated to 95° C. for ˜10 minutes. Samples were cooledto room temperature before being loaded onto SDS polyacrylamide gels orwere stored at −20° C. if not run immediately. Samples were run onprecast 15% polyacrylamide “Ready Gels” (Bio-Rad, Hercules Calif.) usinga Ready Gel Cell electrophoresis apparatus (Bio-Rad) according to thevendor protocols. Typically gels were run at 200 volts for ˜35-45minutes. Gels were stained with Coomassie Blue or were analyzed byWestern Blot following electro-blotting. Coomassie staining of wholecell lysates from strains BOB 129, BOB 133, BOB 121 and BOB 132 showed aband of ˜22 kD that co-migrated with purified recombinant human GHstandard purchased from Research Diagnostics Inc (Flanders, N.J.). Thatband was most prominent in induced cultures following overnightinduction. However, a band was also observed at that molecular weight inuninduced cultures of these same strains and could also be observed withand without induction in the BOB 119 and BOB 130 control strains thatcarried the expression vector pCYB1 lacking the GH gene. To clarify thisobservation, Western Blot analyses were performed on whole cell lysatesof induced cultures of strains BOB119, BOB130, BOB129, BOB133, BOB121,and BOB132. Western blots were performed with polyclonal rabbitanti-human GH antiserum purchased from United States Biological;catalogue # G 9000-11 (Swampscott, Mass.) This primary antibody was usedat a 1:5000 dilution and its binding was detected with goat anti-rabbitIgG Fc conjugated to alkaline phosphatase, (product #31341) purchasedfrom Pierce (Rockford, Ill.). This secondary antibody was used at a1:10,0000 dilution. Alkaline phosphatase activity was detected using theImmunoPure® Fast Red TR/AS-MX Substrate Kit (Pierce, Rockford Ill.)according to the vendor protocols. The Western Blots clearlydemonstrated presence of GH in lysates of induced cultures of BOB129,BOB133, BOB121, and BOB132 at both 3 and 16 h post-induction. In theinduced culture of control strains, BOB119 and BOB130, no GH wasdetected by Western blot at 3 or 16 h post-induction time points.

In these preliminary experiments, the highest yields of GH were obtainedfrom BOB132 W3110 (pBBT114) in which the GH gene is fused downstream ofthe stII secretion signal sequence. This strain was tested further todetermine if the GH protein was secreted to the periplasm as would beexpected. An induced culture of BOB132 was prepared as described aboveand subjected to osmotic shock according to the procedure of Koshlandand Botstein (Cell 20 (1980) pp. 749-760). This procedure ruptures theouter membrane and releases the contents of the periplasm into thesurrounding medium. Subsequent centrifugation separates the periplasmiccontents, present in the supernatant from the remainder of thecell-associated components. In this experiment, the bulk of the GHsynthesized by BOB132 was found to be localized to the periplasm. Thisresult is consistent with the finding that the bulk of the total GH isalso indistinguishable in size from the purified GH standard, whichindicated that the stII signal sequence had been removed. This isindicative of secretion. A larger scale (500 ml) culture of BOB132 wasalso induced, cultured overnight and subjected to osmotic shockaccording to the procedure described by Hsiung et al, 1986(Bio/Technology 4, pp. 991-995). Gel analysis again demonstrated thatthe bulk of the GH produced was soluble, periplasmic, andindistinguishable in size from the GH standard. This material could alsobe quantitatively bound to, and eluted from, a Q-Sepharose column usingconditions very similar to those described for recombinant human GH byBecker and Hsiung, 1986 (FEBS Lett 204 pp145-150).

7. Cloning the Human GH Receptor

The human GH receptor was cloned by PCR using forward primer BB3 andreverse primer BB4. BB3 has the sequence: (SEQ ID NO: 26)5′-CCCCGGATCCGCCACCATGGATCTCTGGCAGCTGCTGTT-3′.

BB4 has the sequence: (SEQ ID NO: 27)5′CCCCGTCGACTCTAGAGCTATTAAATACGTAGCTCTTGGG-3′.The template was single-stranded cDNA prepared from human liver(commercially available from CLONTECH Laboratories). Primers BB3 and BB4contain BamHI and SalI restriction sites, respectively, for cloningpurposes. The 100 μl PCR reactions contained 2.5 ng of thesingle-stranded cDNA and 20 picomoles of each primer in 1×PCR buffer(Perkin-Elmer buffer containing MgCl₂), 200 micromolar concentration ofeach of the four nucleotides dA, dC, dG and dT, 2.5 units of Taqpolymerase (Perkin-Elmer) and 2.5 units of Pfu polymerase (Stratagene,Inc). The PCR reaction conditions were: 96° C. for 3 minutes, 35 cyclesof (95° C., 1 minute; 58° C. for 30 seconds; 72° C. for 2 minutes),followed by 10 minutes at 72° C. The thermocycler employed was theAmplitron II Thermal Cycler (Thermolyne). The approximate 1.9 kb PCRproduct was digested with BamHI and SalI and ligated with similarly cutplasmid pUC19 (New England BioLabs). However, none of the transformantsobtained from this ligation reaction contained the 1.9 kb PCR fragment.Leung et al (Nature 1987 330 pp 537-543) also failed to obtainfull-length cDNA clones of the human GH receptor in pUC19. Subsequentlythe PCR fragment was cloned into a low copy number vector, pACYC184 (NewEngland BioLabs) at the BamHI and SalI sites in this vector. Such cloneswere obtained at reasonable frequencies but E coli strains carrying thecloned PCR fragment grew poorly, forming tiny and heterogeneous lookingcolonies, in the presence of chloramphenicol, which is used to selectfor maintenance of pACYC184.

The PCR fragment was simultaneously cloned into pCDNA3.1 (+)(Invitrogen). The approximate 1.9 kb PCR product was digested with BamHIand SalI and ligated into the

BamHI and XhoI cloning sites of pCDNA3.1 (+). Only infrequenttransformants from this ligation contained the cloned GH receptor cDNAand all of those were found to contain deletions of segments of thereceptor coding sequence. One of these clones was sequenced and found tocontain a deletion of 135 bp within the GH receptor coding sequence: thesequence of the rest of the gene was in agreement with that reported byLeung et at (1987).

8. Cloning the Rabbit GH Receptor

The rabbit GH receptor was cloned by PCR using forward primer BB3(described above) and reverse primer BB36. BB36 has the sequence5′CCCCGTCGACTCTAGAGCCATTAGATACAAAGCTCTTGGG3′ (SEQ ID NO: 41) andcontains XbaI and Sal I restriction sites for cloning purposes. Rabbitliver poly(A)⁺ mRNA was purchased from CLONTECH, Inc. and used as thesubstrate in first strand synthesis of single-stranded cDNA to producetemplate for PCR amplification. First strand synthesis ofsingle-stranded cDNA was accomplished using a 1st Strand cDNA SynthesisKit for RT-PCR (AMV) kit from Boehringer Mannheim Corp (Indianapolis,Ind.) according to the vendor protocols. Parallel first strand cDNAsyntheses were performed using random hexamers or BB36 as the primer.Subsequent PCR reactions with the products of the first strand synthesesas templates, were carried out with primers BB3 and BB36 according tothe 1st Strand cDNA Synthesis Kit for RT-PCR (AMV) kit protocol andusing 2.5 units of Amplitac DNA Polymerase (Perkin-Elmer) and 0.625units of Pfu DNA Polymerase (Stratagene). The PCR reaction conditionswere 96° C. for 3 minutes, 35 cycles of (95° C., 1 minute; 58° C. for 30seconds; 72° C. for 2 minutes), followed by 10 minutes at 72° C. Thethermocycler employed was the Amplitron II Thermal Cycler (Thermolyne).The expected ˜1.9 kb PCR product was observed in PCR reactions usingrandom hexamer-primed or BB36 primed cDNA as template. The randomhexamer-primed cDNA was used in subsequent cloning experiments. It wasdigested with Bam H1 and XbaI and run out over a 1.2% agarose gel. Thisdigest generates two fragments (˜365 bp and ˜1600 bp) because the rabbitGH receptor gene contains an internal Bam H1 site. Both fragments weregel-purified. Initially the ˜1600 bp Bam H1 XbaI fragment was clonedinto pCDNA3.1(+) which had been digested with these same two enzymes.These clones were readily obtained at reasonable frequencies and showedno evidence of deletions as determined by restriction digests andsubsequent sequencing. To generate a full length clone, one of theplasmids containing the 1600 bp Bam H1-Xba I fragment(pCDNA3.1(+)::rab-ghr-2A) was digested with Bam H1, treated with CalfIntestinal Alkaline Phosphatase (Promega) according to the vendorprotocols, gel-purified and ligated with the gel purified ˜365 bp Bam H1fragment that contains the 5′ portion of the rabbit GH receptor gene.Transformants from this ligation were picked and analyzed by restrictiondigestion and PCR to confirm the presence of the ˜365 bp fragment and todetermine its orientation relative to the distal segment of the rabbitGH receptor gene. Three out of four clones analyzed were found tocontain the ˜365 bp fragment cloned in the correct orientation forreconstitution of the rabbit GH receptor gene. The lack of complicationsin the cloning in E coli of the rabbit gene, in contrast to the humangene, is consistent with the results of Leung et al (1987) who alsoreadily obtained full length cDNA clones for the rabbit GH receptor genebut were unable to clone a full length cDNA of the human gene in E coli.The rabbit GH receptor can be employed in assays with human GH as aligand as it has been shown that the human GH binds the rabbit receptorwith high affinity (Leung et al 1987). Plasmids containing the clonedrabbit GH receptor should be sequenced to identify a rabbit GH receptorcDNA with the correct sequence before use.

9. Construction of a Human/Rabbit Chimeric GH Receptor Gene

As an alternative to the rabbit receptor, a chimeric receptor could beconstructed which combines the extracellular domain of the humanreceptor with the transmembrane and cytoplasmic domains of the rabbitreceptor. Such a chimeric receptor could be constructed by recombiningthe human and rabbit genes at the unique Nco I site that is present ineach (Leung et al 1987). Such a recombinant, containing the human genesegment located 5′ to, or “upstream” of the Nco I site and the rabbitgene segment 3′ to, or “downstream” of the Nco I site would encode achimeric receptor of precisely the desired type, having theextracellular domain of the human receptor with the transmembrane andcytoplasmic domains of the rabbit receptor. This would allow analysis ofthe interaction of GH, GH muteins, and PEGylated GH muteins with thenatural receptor binding site but could avoid the necessity of cloningthe full length human GH receptor in E coli.

The GH muteins can be expressed in a variety of expression systems suchas bacteria, yeast or insect cells. Vectors for expressing GH muteins inthese systems are available commercially from a number of suppliers suchas Novagen, Inc. (pET15b for expression in E. coli), New England Biolabs(pC4B1 for expression in E. coli) Invitrogen (pVL1392, pVL1393, andpMELBAC for expression in insect cells using Baculovirus vectors, Pichiavectors for expression in yeast cells, and pCDNA3 for expression inmammalian cells). GH has been successfully produced in E. coli as acytoplasmic protein and as a secreted, periplasmic, protein using the E.coli OmpA or STII signal sequences to promote secretion of the proteininto the periplasmic space (Chang et al., 1987; Hsiung et al., 1986). Itis preferable that the GH muteins are expressed as secreted proteins sothat they do not contain an N-terminal methionine residue, which is notpresent in the natural human protein. For expression in E. coli, DNAsequences encoding GH or GH muteins can be cloned into E. coliexpression vectors such as pET15b that uses the strong T7 promoter orpCYB1 that uses the TAC promoter. Adding IPTG(isopropylthiogalactopyranoside, available form Sigma Chemical Company)to the growth media, can induce expression of the protein. RecombinantGH will be secreted into the periplasmic space from which it can bereleased by, and subsequently purified, following osmotic shock (Beckerand Hsiung, 1986). The protein can be purified further using otherchromatographic methods such as ion-exchange, hydrophobic interaction,size-exclusion and reversed phase chromatography, all of which are wellknown to those of skill in the art (e.g., see Becker and Hsiung, 1986).Protein concentrations can be determined using commercially availableprotein assay kits such as those sold by BioRad Laboratories (Richmond,Calif.). If the GH proteins are insoluble when expressed in E. coli theycan be refolded using procedures well known to those skilled in the art(see Cox et al., 1994 and World patent applications WO9422466 andWO9412219).

Alternatively, the proteins can be expressed in insect cells as secretedproteins. The expression plasmid can be modified to contain the GHsignal sequence to promote secretion of the protein into the medium. ThecDNAs can be cloned into commercially available vectors, e.g., pVL1392from Invitrogen, Inc., and used to infect insect cells. The GH and GHmuteins can be purified from conditioned media using conventionalchromatography procedures. Antibodies to rhGH can be used in conjunctionwith Western blots to localize fractions containing the GH proteinsduring chromatography. Alternatively, fractions containing GH can beidentified using ELISA assays.

The cysteine-added GH variants also can be expressed as intracellular orsecreted proteins in eukaryotic cells such as yeast, insect cells ormammalian cells. Vectors for expressing the proteins and methods forperforming such experiments are described in catalogues from variouscommercial supply companies such as Invitrogen, Inc., Stratagene, Inc.and ClonTech, Inc. The GH and GH muteins can be purified usingconventional chromatography procedures.

Biological activity of the GH muteins can be measured using a cell linethat proliferates in response to GH. Fuh et al. (1992) created aGH-responsive cell line by stably transforming a myeloid leukemia cellline, FDC-P1, with a chimeric receptor comprising the extracellulardomain of the rabbit GH receptor fused to the mouse G-CSF receptor. Thiscell line proliferates in response to GH with a half maximal effectiveconcentration (EC₅₀) of 20 picomolar. A similar cell line can beconstructed using the published sequences of these receptors andstandard molecular biology techniques (Fuh et al., 1992). Alternatively,the extracellular domain of the human GH receptor can be fused to themouse G-CSF receptor using the published sequences of these receptorsand standard molecular biology techniques. Transformed cells expressingthe chimeric receptor can be identified by flow cytometry using labeledGH, by the ability of transformed cells to bind radiolabeled GH, or bythe ability of transformed cells to proliferate in response to added GH.Purified GH and GH muteins can be tested in cell proliferation assaysusing cells expressing the chimeric receptor to measure specificactivities of the proteins. Cells can be plated in 96-well dishes withvarious concentrations of GH or GH muteins. After 18 h, cells aretreated for 4 hours with ³H-thymidine and harvested for determination ofincorporated radioactivity. The EC₅₀ can be determined for each mutein.Assays should be performed at least three times for each mutein usingtriplicate wells for each data point. GH muteins displaying similaroptimal levels of stimulation and EC₅₀ values comparable to or greaterthan wild type GH are preferable.

GH muteins that retain in vitro activity can be PEGylated using acysteine-reactive 8 kDa PEG-maleimide (or PEG-vinylsulfone) commerciallyavailable from Shearwater, Inc. Generally, methods for PEGylating theproteins with these reagents will be similar to those described in worldpatent applications WO 9412219 and WO 9422466 and PCT applicationUS95/06540, with minor modifications. The recombinant proteins must bepartially reduced with dithiothreitol (DTT) in order to achieve optimalPEGylation of the free cysteine. Although the free cysteine is notinvolved in a disulfide bond, it is relatively unreactive tocysteine-reactive PEGs unless this partial reduction step is performed.The amount of DTT required to partially reduce each mutein can bedetermined empirically, using a range of DTT concentrations. Typically,a 5-10 fold molar excess of DTT for 30 min at room temperature issufficient. Partial reduction can be detected by a slight shift in theelution profile of the protein from a reversed-phase column. Care mustbe taken not to over-reduce the protein and expose additional cysteineresidues. Over-reduction can be detected by reversed phase-HPLC (theprotein will have a retention time similar to the fully reduced anddenatured protein) and by the appearance of GH molecules containing twoPEGs (detectable by a molecular weight change on SDS-PAGE). Wild type GHcan serve as a control since it should not PEGylate under similarconditions. Excess DTT can be removed by size exclusion chromatographyusing spin columns. The partially reduced protein can be reacted withvarious concentrations of PEG-maleimide (PEG: protein molar ratios of1:1, 5:1, 10:1 and 50:1) to determine the optimum ratio of the tworeagents. PEGylation of the protein can be monitored by a molecularweight shift using sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE). The lowest amount of PEG that givessignificant quantities of mono-pegylated product without givingdi-pegylated product will be considered optimum (80% conversion tomono-pegylated product is considered good). Generally, mono-PEGylatedprotein can be purified from non-PEGylated protein and unreacted PEG bysize-exclusion or ion exchange chromatography. The purified PEGylatedprotein can be tested in the cell proliferation assay described above todetermine its specific activity.

The above experiments will allow identification of amino acids in theB-C loop, C-D loop or N-terminal end of the A-B loop in GH that can bechanged to cysteine residues, PEGylated and retain in vitro biologicalactivity. These muteins can be tested in animal disease models wellknown in the art.

Experiments can be performed to confirm that the PEG molecule isattached to the protein at the proper site. This can be accomplished byproteolytic digestion of the protein, purification of the PEG peptide(which will have a large molecular weight) by size exclusion, ionexchange or reversed phase chromatography, followed by amino acidsequencing or mass spectroscopy. The PEG-coupled amino acid will appearas a blank in the amino acid sequencing run.

The pharmacokinetic properties of the PEG-GH proteins can be determinedas follows, or as described in world patent application WO9422466. Pairsof rats or mice can receive an intravenous bolus injection of the testproteins. Circulating levels of the proteins are measured over thecourse of 24 h by removing a small sample of blood from the animals atdesired time points. Circulating levels of the test proteins can bequantitated using ELISA assays. Additional experiments can be performedusing the subcutaneous route to administer the proteins. Similarexperiments should be performed with the non-PEGylated protein to serveas a control. These experiments will reveal whether attachment of a PEGreagent to the protein alters its pharmacokinetic properties. Covalentmodification of the protein with PEG should increase the protein'scirculating half-life relative to the unPEGylated protein. Larger PEGmolecules and/or attachment of multiple PEG molecules should lengthenthe circulating half-life longer than smaller PEG molecules.

PEG-GH proteins can be tested in rodent models of growth hormonedeficiency (Cox et al., 1994) and cachexia (Tomas et al., 1992; Read etal., 1992) to determine optimum dosing schedules and demonstrateefficacy. These studies can explore different size PEG molecules, e.g.,8 and 20 kDa, and dosing schedules to determine the optimum PEG size anddosing schedule. It is expected that the larger PEG molecule willincrease the circulating half-life greater than the smaller PEG moleculeand will require less frequent dosing. However, large proteinspotentially may have reduced volumes of distribution in vivo; thus, itis possible a 20 kDa PEG attached to GH will limit bioavailability,reducing its efficacy. Rodent models will allow determination of whetherthis is the case. Once the optimum dosing schedules and PEG sizes aredetermined, the efficacy of PEG-GH to GH can be compared in the animalmodels. While all PEG-GH proteins having GH activity are included in theinvention, the preferred PEG-GH proteins are those that enhance growthequal or superior to GH, but which can be given less frequently. PEG-GHshould be more efficacious than GH when both are administered using theless frequent dosing schedules.

One GH deficiency model that can be used is a hypophysectomized rat. GHstimulates body weight gain and bone and cartilage growth in this model(Cox et al., 1994). Hypophysectomized rats can be purchased from CharlesRiver. Rats can be injected with GH, PEG-GH or placebo and weight gainmeasured daily over a 10-14 day period. At time of sacrifice, tibialepiphysis width can be determined as a measure of bone growth.Experimental methods for performing these studies are described in Coxet al. (1994).

The efficacy of PEG-GH in rodent cachexia models can be tested in asimilar manner. Daily administration of dexamethasone, via osmotic pumpsor subcutaneous injection, can be used to induce weight loss (Tomas etal., 1992; Read et al., 1992; PCT patent application US95/06540).

EXAMPLE 2 Cysteine-Added Variants of Erythropoietin

This example relates to cysteine-added variants of erythropoietin (EPO).EPO is the hormone primarily responsible for stimulating erythropoiesisor red blood cell formation. EPO acts on immature red blood cellprecursors to stimulate their further proliferation and differentiationinto mature red blood cells. A commercial pharmaceutical version isavailable from Amgen, Inc. Human EPO is a 35-39 kDa glycoproteinsecreted by the adult kidney. The mature human protein contains 166amino acids and is heavily glycosylated. The sequence of human EPO (SEQID NO: 2) is shown in Lin et al 1985 and Jacobs et al. 1985, which areincorporated herein by reference. The primary sequence of EPO is highlyconserved among species (greater than 80% identity; Wen et al., 1994).Sugar groups account for greater than 40% of the protein's mass. HumanEPO contains three N-linked glycosylation sites and one O-linkedglycosylation site. The N-linked glycosylation sites are conserved indifferent species whereas the O-linked glycosylation site is not. Theextensive glycosylation of EPO has prevented the protein'scrystallization, so the X-ray structure of the protein is not known.Human EPO contains four cysteine residues. The disulfide assignments areCys7 to Cys161 and Cys29 to Cys33. Cys33 is not conserved in mouse EPO,suggesting that the Cys29-Cys33 disulfide bond is not critical to mouseEPO's structure or function. This conclusion also seems to hold forhuman EPO (Boissel et al., 1993).

The amino acid sequence of EPO is consistent with the protein being amember of the GH supergene family and mutational studies support thisview of EPO's structure (Boissel et al., 1993; Wen et al., 1994). Amodel of the three dimensional structure of EPO, modeled after the GHstructure has been proposed (Boissel et al., 1993; Wen et al., 1994).Helix A encompasses amino acids 9-22, Helix B encompasses amino acids59-76, Helix C encompasses amino acids 90-107 and Helix D encompassesamino acids 132-152. Amino acids in EPO important for receptor bindinghave been identified through mutagenesis experiments and resideprimarily in the N-terminal half of presumptive helix A and theC-terminal half of presumptive helix D (Boissel et al., 1993; Wen etal., 1994; Matthews et al., 1996). Only a single cell surface receptorfor EPO has been identified (D'Andrea et al., 1989). It is believed thatEPO dimerizes its receptor in much the same way that GH dimerizes itsreceptor (Matthew's et al., 1996).

Human EPO contains three sites for N-linked glycosylation(asparagine-24, -38 and -83) and one site for O-linked glycosylation(serine-126). The N-linked glycosylation sites are located in the A-Band B-C loops and the O-glycosylation site is located in the C-D loop.The N-linked glycosylation sites are conserved among species whereas theO-linked glycosylation site is absent in rodent EPO (Wen et al., 1993).A non-O-linked glycosylated human variant containing methionine atposition 126 has been described (U.S. Pat. No. 4,703,008). The N-linkedsugar groups are heavily branched and contain terminal sialic acidresidues (Sasaki et al., 1987; Takeuchi et al., 1988). N-38 and N-83contain the most highly branched oligosaccharides (Sasaki et al., 1988).

The terminal sialic residues on EPO are critical for the protein's invivo function because removal of these residues by digestion eliminatesin vivo activity (Fukada et al., 1989; Spivak and Hogans, 1989). Loss ofactivity correlates with faster clearance of the asialated protein fromthe body. The circulating half-life of the asialated protein in rats isless than ten minutes, in contrast to that of the sialated protein,which is approximately 2 hr (Fukada et al., 1989; Spivak and Hogans,1989). Thus, in vivo activity of EPO directly correlates with itscirculating half-life.

The role of N-linked sugars in EPO's biological activities has beenbetter defined by mutating, individually and in combination, the threeasparagine residues comprising the N-linked glycosylation sites. EPOmuteins in which only a single N-linked glycosylation site was mutated,i.e., N24Q, N38Q and N83Q, were secreted from mammalian cells asefficiently as wild type EPO, indicating that N-linked glycosylation atall three sites is not required for protein secretion (N24Q indicatesthat the asparagine at position 24 is mutated to glutamine). Incontrast, EPO muteins in which two or more N-linked glycosylation siteswere mutated were secreted less efficiently than wild type EPO frommammalian cells (Yamaguchi et al., 1991; Delorme et al., 1992).Mutagenesis studies found that each of the single N-linked glycosylationsite muteins had in vitro biological activities equal to or greater thanwild type EPO. Thus, it was concluded that none of the N-linkedglycosylation sites is essential for secretion or in vitro biologicalactivity of EPO. In fact, removal of one of the glycosylation sitesseemed to improve biological activity (Yamaguchi et al., 1991).

The in vivo biological activity of N-linked glycosylation muteins wasstudied by two groups. Yamaguchi et al. (1991) concluded that the N24Qand N83Q muteins had in vivo activities greater than wild type EPO,which correlated with their increased in vitro activities. These authorsfound that the N38Q mutein had decreased in vivo activity, about 60% ofwild type EPO. N38 is the most heavily branched of the three N-linkedglycosylation sites (Sasaki et al., 1988). Delorme et al. (1992)reported that mutating any of the N-linked glycosylation sites reducedin vivo biological activity by about 50%. Muteins in which two or moreglycosylation sites were mutated had decreased in vivo activities inboth studies.

The above studies indicate that some N-linked glycosylation is requiredfor in vitro and in vivo activity of EPO. Individually, however, none ofthe three glycosylation sites is absolutely essential for activity. TheN-linked sugars increase the apparent molecular weight of EPO andprolong its circulating half-life, which correlates with bioactivity.Natural EPO and EPO manufactured in mammalian cells have complexN-linked sugars containing galactose and terminal sialic acid residues.The galactose residues are recognized by specific receptors onhepatocytes and promote rapid clearance of EPO from the body unless thegalactose residues are masked by the terminal sialic acid residues.

Mutagenesis studies concluded that O-linked glycosylation is notrequired for in vitro or in vivo function of EPO (Delorme et al., 1992).This is in keeping with the observation that rodent EPO is notO-glycosylated and with the existence of a naturally occurring human EPOvariant in which serine-126 is replaced by methionine, with acorresponding lack of O-linked glycosylation. Mutagenesis of serine-126revealed that certain amino acid changes at this site (to valine,histidine or glutamic acid) yielded EPO muteins with biologicalactivities similar to wild type EPO, whereas other amino acid changes(to alanine or glycine) resulted in EPO molecules with severely reducedactivities (Delorme et al., 1992). The effect of changing serine-126 tocysteine was not studied. The in vivo bioactivity of S126V EPO was foundto be similar to wild type EPO (Delorme et al., 1992).

The requirement for complex, N-linked carbohydrates containing terminalsialic acid residues for in vivo activity of EPO has limited commercialmanufacture of the protein to mammalian cells. The important functionsof the sialated N-linked sugars are to prevent protein aggregation,increase protein stability and prolong the circulating half-life of theprotein. The terminal sialic acid residues prolong EPO's circulatinghalf-life by masking the underlying galactose residues, which arerecognized by specific receptors on hepatocytes and promote clearance ofthe asialated protein. EPO can be produced in insect cells and isN-glycosylated and fully active in vitro; its activity in vivo has notbeen reported (Wojchowski et al., 1987).

This example provides for the design of cysteine-added EPO variants andtheir use in preparing conjugates using cysteine-reactive PEGs and othercysteine-reactive moieties. Certain amino acids in EPO are non-essentialfor biological activity and can be mutated to cysteine residues withoutaltering the normal disulfide binding pattern and overall conformationof the molecule. These amino acids are located in the A-B loop (aminoacids 23-58 of the mature protein sequence), the B-C loop (amino acids77-89 of the mature protein sequence), the C-D loop (amino acids 108-131of the mature protein sequence), proximal to helix A (amino acids 1-8)and distal to helix D (amino acids 153-166 of the mature proteinsequence). Also contemplated as preferred sites for adding cysteineresidues are at the N-terminus or C-terminus of the protein sequence.Preferred sites for cysteine substitutions are the O-linkedglycosylation site (serine-126) and the amino acids comprising the threeN-linked glycosylation sites (N24, I25, T26, N38, I39, T40, N83, S84,S85). Glycosylation sites are attractive sites for introducing cysteinesubstitutions and attaching PEG molecules to EPO because (1) these sitesare surface exposed; (2) the natural protein can tolerate bulky sugargroups at these positions; (3) the glycosylation sites are located inthe putative loop regions and away from the receptor binding site (Wenet al., 1994); and (4) mutagenesis studies indicate these sites (atleast individually) are not essential for in vitro or in vivo activity(Yamaguchi et al., 1991; Delorme et al., 1992). As discussed above, thelocal conformation of the region encompassing the O-glycosylation siteregion seems to be important for biological activity. Whether a cysteinesubstitution at position 126 affects biological activity has not beenstudied. The cysteine-29 to cysteine-33 disulfide bond is not necessaryfor biological activity of EPO because changing both residues totyrosine simultaneously yielded a biologically active EPO protein(Boissel et al., 1993; Wen et al., 1994). A “free” cysteine can becreated by changing either cysteine-29 or cysteine-33 to another aminoacid. Preferred amino acid changes would be to serine or alanine. Theremaining “free” cysteine (cysteine-29 or cysteine-33) would be apreferred site for covalently modifying the protein withcysteine-reactive moieties.

Bill et al. (1995) individually substituted cysteine for N24, N38 andN83 and reported that the muteins had greatly reduced in vitrobiological activities (less than 20% of wild type activity). Bill et al.(1995) expressed the EPO variants as fusion proteins (fused toglutathionine-5-transferase) in bacteria. One aspect of the presentinvention is to provide expression systems in which the N24C, N38C andN83C EPO variants will have in vitro biological activities more similarto wild type EPO.

U.S. Pat. No. 4,703,008 contemplates naturally occurring variants of EPOas well as amino acid substitutions that are present in EPO proteins ofmammals. Ovine EPO contains a cysteine residue at position 88 of thepolypeptide chain. The inventor is unaware of any other naturallyoccurring human or animal cysteine variants of EPO in which the cysteineresidue occurs in the polypeptide regions disclosed herein as beinguseful for generating cysteine-added EPO variants. U.S. Pat. No.4,703,008 specifically teaches away from cysteine-added EPO variants bysuggesting that expression of EPO might be improved by deleting cysteineresidues or substituting naturally occurring cysteine residues withserine or histidine residues.

The mature protein form of EPO can contain 165 or 166 amino acidsbecause of post-translational removal of the C-terminal arginine.Asp-165 is the C-terminus of the 165 amino acid form and Agr-166 is theC-terminal amino acid of the 166 amino acid form. The cysteinesubstitution and insertion mutations described herein can compriseeither the 165 or 166 amino acid forms of mature EPO.

A cDNA encoding EPO can be cloned using the polymerase chain reaction(PCR) technique from the human HepG2 or Hep3B cell lines, which areknown to express EPO when treated with hypoxia or cobalt chloride (Wenet al., 1993) and are available from the American Type CultureCollection (ATCC). Cysteine mutations can be introduced into the cDNA bystandard phage, plasmid or PCR mutagenesis procedures as described forGH. As described above, the preferred sites for introduction of cysteinesubstitution mutations are in the A-B loop, the B-C loop, the C-D loopand the region proximal to helix A and distal to helix D. The mostpreferred sites in these regions are the N- and O-linked glycosylationsites: S126C; N24C; I25C, T26C; N38C; I39C, T40C; N83C, S84C and S85C.Other preferred sites for cysteine substitution mutagenesis are in theA-B loop, the B-C loop and the C-D loop, amino acids surrounding theglycosylation sites and the region of the protein proximal to helix Aand distal to helix D (Boissel et al., 1993; Wen et al., 1994). Otherpreferred sites for cysteine substitutions in these regions are: A1, P2,P3, R4, D8, S9, T27, G28, A30, E31, H32, S34, N36, D43, T44, K45, N47,A50, K52, E55, G57, Q58, G77, Q78, A79, Q86, W88, E89, T107, R110, A111,G113, A114, Q115, K116, E117, A118, S120, P121, P122, D123, A124, A125,A127, A128, T132, K154, T157, G158, E159, A160, T163, G164, D165 andR166. Cysteine residues also can be introduced proximal to the firstamino acid of the mature protein, i.e., proximal to A1, or distal to thefinal amino acid in the mature protein, i.e., distal to D165 or R166.Other variants in which cys-29 or cys-33 have been replaced with otheramino acids, preferably serine or alanine, also are provided.

Wild type EPO and EPO muteins can be expressed using insect cells todetermine whether the cysteine-added muteins are biologically active.DNAs encoding EPO/EPO muteins can be cloned into the Baculovirusexpression vector pVL1392 (available from Invitrogen, Inc. and SigmaCorporation (St. Louis, Mo.) and used to infect insect cells.Recombinant Baculoviruses producing EPO can be identified by Westernblots of infected insect cell conditioned media using polyclonalanti-human EPO antiserum (available from R&D Systems). The secreted EPOmutein proteins can be purified by conventional chromatographicprocedures well known to those of skill in the art. Proteinconcentrations can be determined using commercially available proteinassay kits or ELISA assay kits (available from R&D Systems and Bio-RadLaboratories).

Purified EPO and EPO muteins can be tested in cell proliferation assaysusing EPO-responsive cell lines such as UT7-epo (Wen et al., 1994) orTF1 (available from the ATCC) to measure specific activities of theproteins. Cells can be plated in 96-well microtiter plates with variousconcentrations of EPO. Assays should be performed in triplicate. After1-3 days in culture, cell proliferation can be measured by ³H-thymidineincorporation, as described above for GH. The concentration of proteingiving half-maximal stimulation (EC₅₀) can be determined for eachmutein. Assays should be performed at least three times for each mutein,with triplicate wells for each data point. EC₅₀ values can be used tocompare the relative potencies of the muteins. Alternatively, cellproliferation in response to added EPO muteins can be analyzed using anMTT dye-exclusion assay (Komatsu et al. 1991). Proteins displayingsimilar optimal levels of stimulation and EC₅₀ values comparable to orgreater than wild type EPO are preferable.

The above studies confirm identification of amino acid residues in EPOthat can be changed to cysteine residues and retain biological activity.Muteins that retain activity can be PEGylated using a cysteine-reactive8 kDa PEG-maleimide as described above for GH muteins. Wild type EPOshould be used as a control since it should not react with thecysteine-reactive PEG under identical partial reduction conditions. Thelowest amount of PEG that gives significant quantities of mono-PEGylatedproduct without giving di-PEGylated product should be consideredoptimum. Mono-PEGylated protein can be purified from non-PEGylatedprotein and unreacted PEG by size-exclusion or ion exchangechromatography. The purified PEGylated proteins should be tested in thecell proliferation assay described above to determine theirbioactivities.

One or more of the PEGylated EPO muteins that retain in vitrobioactivity, are candidates for testing in animal disease models.PEGylation of the protein at the proper amino acid can be determined asdescribed for GH.

In vivo testing of PEGylated EPO muteins expressed using insect cellsmay require that they be re-engineered for expression in mammalian cellsto ensure proper glycosylation. PEG-EPO candidates produced using insectcells can be tested in the animal models described below to determine ifthey are active in vivo and whether they are as active as PEG-EPOproduced using mammalian cell expression systems. For expression inmammalian cells, the EPO muteins can be subcloned into commerciallyavailable eukaryotic expression vectors and used to stably transformChinese Hamster Ovary (CHO) cells (available from the ATCC). Sublinescan be screened for EPO expression using ELISA assays. Sufficientquantities of the insect cell- and mammalian cell-produced EPO muteinscan be prepared to compare their biological activities in animal anemiamodels.

In vivo bioactivities of the EPO muteins can be tested using theartificial polycythemia or starved rodent models (Cotes and Bangham.,1961; Goldwasser and Gross., 1975). In the starved rodent model, ratsare deprived of food on day one and treated with test samples on daystwo and three. On day four, rats receive an injection of radioactiveiron-59. Approximately 18 h later, rats are anesthetized and bloodsamples drawn. The percent conversion of labeled iron into red bloodcells is then determined. In the artificial polycythemia model, mice aremaintained in a closed tank and exposed for several days to hypobaricair. The animals are then brought to normal air pressure. Red blood cellformation is suppressed for several days. On day four or six afterreturn to normal air pressure, mice are injected with erythropoietin orsaline. Mice receive one injection per day for one to two days. One daylater the animals receive an intravenous injection of labeled iron-59.The mice are euthanized 20 h later and the amount of labeled ironincorporated into red blood cells determined. EPO stimulates red bloodcell formation in both models as measured by a dose-dependent increasein labeled iron incorporated into red blood cells. In both modelsdifferent dosing regimens and different times of injections can bestudied to determine if PEG-EPO is biologically active and/or morepotent and produces longer acting effects than natural EPO.

EXAMPLE 3 Alpha Interferon

Alpha interferon is produced by leukocytes and has antiviral, anti-tumorand immunomodulatory effects. There are at least 20 distinct alphainterferon genes that encode proteins that share 70% or greater aminoacid identity. Amino acid sequences of the known alpha interferonspecies are given in Blatt et al. (1996). A “consensus” interferon thatincorporates the most common amino acids into a single polypeptide chainhas been described (Blatt et al., 1996). A hybrid alpha interferonprotein may be produced by splicing different parts of alpha interferonproteins into a single protein (Horisberger and Di Marco, 1995). Somealpha interferons contain N-linked glycosylation sites in the regionproximal to helix A and near the B-C loop (Blatt et al. 1966). The alpha2 interferon protein (SEQ ID NO: 3) contains four cysteine residues thatform two disulfide bonds. The cys1-cys98 disulfide bond (cys1-cys99 insome alpha interferon species such as alpha 1; SEQ ID NO: 4) is notessential for activity. The alpha 2-interferon protein does not containany N-linked glycosylation sites. There are two subtypes of the alpha-2interferon protein; one subtype has arginine at position 23 whereas theother subtype has lysine at position 23. The crystal structure of alphainterferon has been determined (Radhakrishnan et al., 1996). Alphainterferon has five major alpha helices referred to as helices A-E.Helix A encompasses amino acids 9-21, helix B encompasses amino acids52-68, helix C encompasses amino acids 78-100, helix D encompasses aminoacids 112-132 and helix E encompasses amino acids 137-160.

This example provides cysteine added variants in the region proximal tothe A helix (amino acids 1-8), distal to the E helix (amino acids161-165), in the A-B loop (amino acids 22-51), in the B-C loop (aminoacids 69-77), in the C-D loop (amino acids 101-111) and in the D-E loop(amino acids 133-136). This Example also provides cysteine-addedvariants at the first three or last three amino acids in helices A, B,C, D and E. Preferred sites for the introduction of cysteine residues inthese regions of the alpha interferon-2 species are: D2, L3, P4, Q5, T6,S8, Q20, R22, K/R23, S25, F27, S28, K31, D32, R33, D35, G37, F38, Q40,E41, E42, F43, G44, N45, Q46, F47, Q48, K49, A50, N65, S68, T69, K70,D71, S72, S73, A74, A75, D77, E78, T79, Y89, Q90, Q91, N93, D94, E96,A97, Q101, G102, G104, T106, E107, T108, P109, K112, E113, D114, S115,K131, E132, K133, K134, Y135, S136, A139, S152, S154, T155, N156, L157,Q158, E159, S160, L161, R162, S163, K164, E165. Variants in whichcysteine residues are introduced proximal to the first amino acid of themature protein, i.e., proximal to C1, or distal to the final amino acidin the mature protein, i.e., distal to E165 are provided. Other variantsin which cys-1 or cys-98 (cys-99 in some alpha interferon species) havebeen replaced with other amino acids, preferably serine or alanine, alsoare provided. Other variants in which Cys-1 has been deleted (des Cys-1)also are provided. The cysteine variants may be in the context of anynaturally occurring or non-natural alpha interferon sequence, e.g.,consensus interferon or interferon protein hybrids. Some naturallyoccurring alpha interferon species (e.g., alpha interferon-1) contain anaturally occurring “free” cysteine. In such interferon species thenaturally occurring free cysteine can be changed to another amino acid,preferably serine or alanine.

This example also provides cysteine variants of other alpha interferonspecies, including consensus interferon, at equivalent sites in theseproteins. The alignment of the alpha interferon-2 species with otherknown alpha interferon species and consensus interferon is given inBlatt et al. (1996). The crystal structure of alpha interferon-2 hasbeen determined by Rhadhakrishnan et al. (1996). Lydon et al (1985)found that deletion of the first four amino acids from the N-terminus ofalpha interferon did not affect biological activity. Valenzuela et al(1985) found that substitution of Phe-47 in alpha interferon-2 with Cys,Tyr or Ser did not alter biological activity of the protein. Cys-1 andCys-98 have been changed individually to glycine and serine,respectively, without altering biological activity of the protein(DeChiara et al, 1986).

DNA sequences encoding alpha interferon-2 can be amplified from humangenomic DNA, since alpha interferon genes do not contain introns (Pestkaet al., 1987). The DNA sequence of alpha interferon-2 is given inGoeddel et al. (1980). Alternatively, a cDNA for alpha interferon-2 canbe isolated from human lymphoblastoid cell lines that are known toexpress alpha interferon spontaneously or after exposure to viruses(Goeddell et al., 1980; Pickering et al., 1980). Many of these celllines are available from the American Type Culture Collection(Rockville, Md.). Specific mutations can be introduced into the alphainterferon sequence using plasmid-based site-directed mutagenesis kits(e.g., Quick-Change Mutagenesis Kit, Stratagene, Inc.), phagemutagenesis strategies or employing PCR mutagenesis as described for GH.

Alpha interferon has been successfully produced in E. coli as anintracellular protein (Tarnowski et al., 1986; Thatcher and Panayotatos,1986). Similar procedures can be used to express alpha interferonmuteins. Plasmids encoding alpha interferon or alpha interferon muteinscan be cloned into an E. coli expression vector such as pET15b(Novagene, Inc.) that uses the strong T7 promoter or pCYB1 (New EnglandBioLabs, Beverly, Mass.) that uses the TAC promoter. Expression of theprotein can be induced by adding IPTG to the growth media.

Recombinant alpha interferon expressed in E. coli is sometimes solubleand sometimes insoluble (Tarnowski et al., 1986; Thatcher andPanayotatos, 1986). Insolubility appears to be related to the degree ofoverexpression of the protein. Insoluble alpha interferon proteins canbe recovered as inclusion bodies and renatured to a fully activeconformation following standard oxidative refolding protocols (Thatcherand Panayotatos, 1986; Cox et al., 1994). The alpha interferon proteinscan be purified further using other chromatographic methods such asion-exchange, hydrophobic interaction, size-exclusion and reversed phaseresins (Thatcher and Panayotaos, 1986). Protein concentrations can bedetermined using commercially available protein assay kits (Bio-RadLaboratories).

If E. coli expression of alpha interferon muteins is not successful, onecan express the proteins in insect cells as secreted proteins asdescribed for GH. The proteins can be modified to contain the naturalalpha interferon signal sequence (Goeddell et al., 1980) or the honeybeemellitin signal sequence (Invitrogen, Inc.) to promote secretion of theproteins. Alpha interferon and alpha interferon muteins can be purifiedfrom conditioned media using conventional chromatography procedures.Antibodies to alpha interferon can be used in conjunction with Westernblots to localize fractions containing the alpha interferon proteinsduring chromatography. Alternatively, fractions containing alphainterferon proteins can be identified using ELISAs.

Bioactivities of alpha interferon and alpha interferon muteins can bemeasured using an in vitro viral plaque reduction assay (Ozes et al.,1992; Lewis, 1995). Human HeLa cells can be plated in 96-well plates andgrown to near confluency at 37° C. The cells are then washed and treatedfor 24 hour with different concentrations of each alpha interferonpreparation. Controls should include no alpha interferon and wild typealpha interferon (commercially available from Endogen, Inc., Woburn,Mass.). A virus such as Vesicular stomatitis virus (VSV) orencephalomyocarditis virus (EMCV) is added to the plates and the platesincubated for a further 24-48 hours at 37° C. Additional controls shouldinclude samples without virus. When 90% or more of the cells have beenkilled in the virus-treated, no alpha interferon control wells(determined by visual inspection of the wells), the cell monolayer arestained with crystal violet and absorbance of the wells read using amicroplate reader. Alternatively, the cell monolayers can be stainedwith the dye MTT (Lewis, 1995). Samples should be analyzed in duplicateor triplicate. EC₅₀ values (the amount of protein required to inhibitthe cytopathic effect of the virus by 50%) can be used to compare therelative potencies of the proteins. Wild type alpha interferon-2protects cells from the cytopathic effects of VSV and EMCV and has aspecific activity of approximately 2×10⁸ units/mg in this assay (Ozes etal., 1992). Alpha interferon muteins displaying EC₅₀ values comparableto wild type Alpha Interferon are preferable.

Alpha interferon muteins that retain activity can be PEGylated usingprocedures similar to those described for GH. Wild type alphainterferon-2 can serve as a control since it should not PEGylate undersimilar conditions. The lowest amount of PEG that gives significantquantities of mono-pegylated product without giving di-pegylated productshould be considered optimum. Mono-PEGylated protein can be purifiedfrom non-PEGylated protein and unreacted PEG by size-exclusion or ionexchange chromatography. The purified PEGylated proteins can be testedin the viral plaque reduction bioassay described above to determinetheir bioactivities. PEGylated alpha interferon proteins withbioactivities comparable to wild type alpha interferon are preferable.Mapping the PEG attachment site and determination of pharmacokineticdata for the PEGylated protein can be performed as described for GH.

In vivo bioactivities of the PEG-alpha interferon muteins can be testedusing tumor xenograft models in nude mice and viral infection models(Balkwill, 1986; Fish et al., 1986). Since PEG-alpha interferonbioactivity may be species-specific, one should confirm activity of thePEGylated protein using appropriate animal cell lines in in vitro virusplaque reduction assays, similar to those described above. Next, oneshould explore the effects of different dosing regimens and differenttimes of injections to determine if PEG-alpha interferon is more potentand produces longer lasting effects than non-PEGylated alpha interferon.

The novel alpha interferon-derived molecules of this example can beformulated and tested for activity essentially as set forth in Examples1 and 2, substituting, however, the appropriate assays and otherconsiderations known in the art related to the specific proteins of thisexample.

EXAMPLE 4 Beta Interferon

Beta interferon is produced by fibroblasts and exhibits antiviral,antitumor and immunomodulatory effects. The single-copy beta interferongene encodes a preprotein that is cleaved to yield a mature protein of166 amino acids (Taniguchi et al. 1980; SEQ ID NO: 5). The proteincontains three cysteines, one of which, cysteine-17, is “free”, i.e., itdoes not participate in a disulfide bond. The protein contains oneN-linked glycosylation site. The crystal structure of the protein hasbeen determined (Karpusas et al. 1997). Beta interferon has five majoralpha helices referred to as helices A-E. Helix A encompasses aminoacids 2-22, helix B encompasses amino acids 51-71, helix C encompassesamino acids 80-107, helix D encompasses amino acids 118-136 and helix Eencompasses amino acids 139-162.

This example provides cysteine-added variants at any of the three aminoacids that comprise the N-linked glycosylation sites, i.e., N80C, E81Cor T82C. This example also provides cysteine-added variants in theregion proximal to the A helix (amino acid 1), distal to the E helix(amino acids 163-166), in the A-B loop (amino acids 23-50), in the B-Cloop (amino acids 72-79), in the C-D loop (amino acids 108-117) and inthe D-E loop (amino acids 137-138). This Example also providescysteine-added variants at the first three or last three amino acids inhelices A, B, C, D and E. Preferred sites for introduction of cysteineresidues in these regions are: M1, S2, Y3, N4, L5, Q23, N25, G26, R27,E29, Y30, K33, D34, R35, N37, D39, E42, E43, K45, Q46, L47, Q48, Q49,Q51, K52, E53, A68, F70, R71, Q72, D73, S74, S75, S76, T77, G78, E107,K108, E109, D110, F111, T112, R113, G114, K115, L116, A135, K136, E137,K138, S139, I157, N158, R159, L160, T161, G162, Y163, L164, R165 andN166. Variants in which cysteine residues are introduced proximal to thefirst amino acid of the mature protein, i.e., proximal to M1, or distalto the final amino acid in the mature protein, i.e., distal to N166 alsoare provided.

These variants are produced in the context of the natural proteinsequence or a variant protein in which the naturally occurring “free”cysteine residue (cysteine-17) has been changed to another amino acid,preferably serine or alanine.

The novel beta interferon-derived molecules of this example can beformulated and tested for activity essentially as set forth in Examples1 and 2, substituting, however, the appropriate assays and otherconsiderations known in the art related to the specific proteins of thisexample.

EXAMPLE 5 Granulocyte Colony-Stimulating Factor (G-CSF)

G-CSF is a pleuripotent cytokine that stimulates the proliferation,differentiation and function of granulocytes. The protein is produced byactivated monocytes and macrophages. The amino acid sequence of G-CSF(SEQ ID NO: 6) is given in Souza et al. (1986), Nagata et al. (1986a, b)and U.S. Pat. No. 4,810,643 all incorporated herein by reference. Thehuman protein is synthesized as a preprotein of 204 or 207 amino acidsthat is cleaved to yield mature proteins of 174 or 177 amino acids. Thelarger form has lower specific activity than the smaller form. Theprotein contains five cysteines, four of which are involved in disulfidebonds. Cysteine-17 is not involved in a disulfide bond. Substitution ofcysteine-17 with serine yields a mutant G-CSF protein that is fullyactive (U.S. Pat. No. 4,810,643). The protein is O-glycosylated atthreonine-133 of the mature protein. G-CSF contains four major alphahelices referred to as helices A-D. Helix A encompasses amino acids11-39, helix B encompasses amino acids 71-91, helix C encompasses aminoacids 100-123 and helix D encompasses amino acids 143-172.

This example provides a cysteine-added variant at threonine-133. Thisexample provides other cysteine-added variants in the region proximal tohelix A (amino acids 1-10), distal to helix D (amino acids 173-174), inthe A-B loop (amino acids 40-70), B-C loop (amino acids 92-99) and C-Dloop (amino acids 124-142). This Example also provides cysteine-addedvariants at the first three or last three amino acids in helices A, B, Cand D. Preferred sites for introduction of cysteine substitutions inthese regions are: T1, P2, L3, G4, P5, A6, S7, S8, L9, P10, Q11, S12,T38, K40, S53, G55, W58, A59, P60, S62, S63, P65, S66, Q67, A68, Q70,A72, Q90, A91, E93, G94, S96, E98, G100, G125, M126, A127, A129, Q131,T133, Q134, G135, A136, A139, A141, S142, A143, Q145, Q173 and P174.Variants in which cysteine residues are introduced proximal to the firstamino acid of the mature protein, i.e., proximal to T1, or distal to thefinal amino acid in the mature protein, i.e., distal to P174 areprovided. These variants are provided in the context of the naturalprotein sequence or a variant protein in which the naturally occurring“free” cysteine residue (cysteine-17) has been changed to another aminoacid, preferably serine or alanine.

A cDNA encoding human G-CSF can be purchased from R&D Systems(Minneapolis, Minn.) or amplified using PCR from mRNA isolated fromhuman carcinoma cell lines such as 5637 and U87-MG known to expressG-CSF constitutively (Park et al., 1989; Nagata, 1994). These cell linesare available from the American Type Culture collection (Rockville,Md.). Specific mutations can be introduced into the G-CSF sequence usingplasmid-based site-directed mutagenesis kits (e.g., Quick-ChangeMutagenesis Kit, Stratagene, Inc.), phage mutagenesis methods oremploying PCR mutagenesis as described for GH.

G-CSF has been successfully produced in E. coli as an intracellularprotein (Souza et al., 1986). One can employ similar procedures toexpress G-CSF and G-CSF muteins. Plasmids encoding G-CSF or G-CSFmuteins can be cloned into an E. coli expression vector such as pET15b(available from Novagen, Inc., Madison, Wis.) that uses the strong T7promoter or pCYB1 (available from New England BioLabs, Beverly, Mass.)that uses the strong TAC promoter. Expression of the protein can beinduced by adding IPTG to the growth media. Recombinant G-CSF expressedin E. coli is insoluble and can be recovered as inclusion bodies. Theprotein can be renatured to a fully active conformation followingstandard oxidative refolding protocols (Souza et al., 1986; Lu et al.,1992; Cox et al., 1994). Similar procedures can be used to refoldcysteine muteins. The proteins can be purified further using otherchromatographic methods such as ion exchange, hydrophobic interaction,size-exclusion and reversed phase resins (Souza et al., 1986; Kuga etal., 1989; Lu et al., 1992). Protein concentrations can be determinedusing commercially available protein assay kits (Bio-Rad Laboratories).

If E. coli expression of G-CSF or G-CSF muteins is not successful, onecan express G-CSF and G-CSF muteins in insect cells as secreted proteinsas described for GH. The proteins can be modified to contain the naturalG-CSF signal sequence (Souza et al., 1986; Nagata et al., 1986a; Nagataet al, 1986b) or the honeybee mellitin signal sequence (Invitrogen,Inc., Carlsbad, Calif.) to promote secretion of the proteins. G-CSF andG-CSF muteins can be purified from conditioned media using conventionalchromatography procedures. Antibodies to G-CSF can be used inconjunction with Western blots to localize fractions containing theG-CSF proteins during chromatography. Alternatively, fractionscontaining G-CSF proteins can be identified using ELISAs.

G-CSF muteins also can be expressed in mammalian cells as described forerythropoietin in Example 2.

Bioactivities of G-CSF and the G-CSF muteins can be measured using an invitro cell proliferation assay. The mouse NFS-60 cell line and the humanAML-193 cell line can be used to measure G-CSF bioactivity (Tsuchiya etal., 1986; Lange et al., 1987; Shirafuji et al., 1989). Both cell linesproliferate in response to human G-CSF. The AML-193 cell line ispreferable since it is of human origin, which eliminates the possibilityof a false conclusion resulting from species differences. The NFS60 celllines proliferates in response to G-CSF with a half-maximal effectiveconcentration (EC₅₀) of 10-20 picomolar. Purified G-CSF and G-CSFmuteins can be tested in cell proliferation assays using these celllines to determine specific activities of the proteins, using publishedmethods (Tsuchiya et al., 1986; Lange et al., 1987; Shirafuji et al.,1989). Cells can be plated in 96-well tissue culture dishes withdifferent concentrations of G-CSF or G-CSF muteins. After 1-3 days at37° C. in a humidified tissue culture incubator, proliferation can bemeasured by ³H-thymidine incorporation as described for GH. Assaysshould be performed at least three times for each mutein usingtriplicate wells for each data point. EC₅₀ values can be used to comparethe relative potencies of the muteins. G-CSF muteins displaying similaroptimal levels of stimulation and EC₅₀ values comparable to wild typeG-CSF are preferable.

G-CSF muteins that retain activity can be PEGylated using proceduressimilar to those described for GH. Wild type G-CSF and ser-17 G-CSF canserve as controls since they should not PEGylate under similarconditions. The lowest amount of PEG that gives significant quantitiesof mono-PEGylated product without giving di-PEGylated product should beconsidered optimum. Mono-PEGylated protein can be purified fromnon-PEGylated protein and unreacted PEG by size-exclusion or ionexchange chromatography. The purified PEGylated proteins can be testedin the cell proliferation assay described above to determine theirbioactivities.

The PEG site in the protein can be mapped using procedures similar tothose described for GH. Pharmacokinetic data for the PEGylated proteinscan be obtained using procedures similar to those described for GH.

Initial studies to demonstrate in vivo efficacy of PEG-G-CSF can be donein normal Sprague-Dawley rats, which can be purchased from CharlesRiver. Groups of rats should receive single subcutaneous or intravenousinjections of various doses of G-CSF, PEG-G-CSF or placebo. Animalsshould be sacrificed at daily intervals for up to a week fordetermination of neutrophil and total white blood cell counts inperipheral blood. Other blood cell types (platelets and red blood cells)can be measured to demonstrate cell specificity.

The efficacy of PEG-G-CSF can be tested in a rat neutropenia model.Neutropenia can be induced by treatment with cyclophosphamide, which isa commonly used chemotherapeutic agent that is myelosuppressive. G-CSFaccelerates recovery of normal neutrophil levels incyclophosphamide-treated animals (Kubota et al., 1990). Rats receive aninjection of cyclophosphamide on day 0 to induce neutropenia. Theanimals are then be divided into different groups, which will receivesubcutaneous injections of G-CSF, PEG-G-CSF or placebo. Neutrophil andtotal white blood cell counts in peripheral blood should be measureddaily until they return to normal levels. Initially one should confirmthat G-CSF accelerates recovery from neutropenia when injected daily.Next, one should explore the effects of different dosing regimens anddifferent times of injections to determine if PEG-G-CSF is more potentand produces longer lasting effects than non-PEGylated G-CSF.

The novel GCSF-derived molecules of this example can be formulated andtested for activity essentially as set forth in Examples 1 and 2,substituting, however, the appropriate assays and other considerationsknown in the art related to the specific proteins of this example.

EXAMPLE 6 Thrombopoietin (TPO)

Thrombopoietin stimulates the development of megakaryocyte precursors ofplatelets. The amino acid sequence of TPO (SEQ ID NO: 7) is given inBartley et al. (1994), Foster et al. (1994), de Sauvage et al. (1994),each incorporated herein by reference.

The protein is synthesized as a 353 amino acid precursor protein that iscleaved to yield a mature protein of 332 amino acids. The N-terminal 154amino acids have homology with EPO and other members of the GH supergenefamily. The C-terminal 199 amino acids do not share homology with anyother known proteins. The C-terminal region contains six N-linkedglycosylation sites and multiple O-linked glycosylation sites (Hoffmanet al., 1996). O-linked glycosylation sites also are found in the regionproximal to the A helix, in the A-B loop, and at the C-terminus of HelixC (Hoffman et al., 1996). A truncated TPO protein containing onlyresidues 1-195 of the mature protein is fully active in vitro (Bartleyet al., 1994).

This example provides cysteine-added variants at any of the amino acidsthat comprise the N-linked glycosylation sites and the O-linkedglycosylation sites. This example also provides cysteine-added variantsin the region proximal to the A helix (amino acids 1-7), distal to the Dhelix (amino acids 148-332), in the A-B loop (amino acids 34-58), in theB-C loop (amino acids 78-86), and in the C-D loop (amino acids 111-122).This Example also provides cysteine-added variants at the first three orlast three amino acids in helices A, B, C and D. Helix a encompassesamino acids 8-33, helix B encompasses amino acids 59-77, helix Cencompasses amino acids 87-110 and helix D encompasses amino acids123-147.

Preferred sites for introduction of cysteine residues are: S1, P2, A3,P4, P5, A6, T37, A43, D45, S47, G49, E50, K52, T53, Q54, E56, E57, T58,A76, A77, R78, G79, Q80, G82, T84, S87, S88, G109, T110, Q111, P113,P114, Q115, G116, R117, T118, T119, A120, H121, K122, G146, G147, S148,T149, A155, T158, T159, A160, S163, T165, S166, T170, N176, R177, T178,S179, G180, E183, T184, N185, F186, T187, A188, S189, A190, T192, T193,G194, S195, N213, Q214, T215, S216, S218, N234, G235, T236, S244, T247,S254, S255, T257, S258, T260, S262, S272, S274, T276, T280, T291, T294,S307, T310, T312, T314, S315, N319, T320, S321, T323, S325, Q326, N327,L328, S329, Q330, E331 and G332. Variants in which cysteine residues areintroduced proximal to the first amino acid of the mature protein, i.e.,proximal to S1, or distal to the final amino acid in the mature protein,i.e., distal to G332 are provided. The cysteine-added variants areprovided in the context of the natural human protein or a variantprotein that is truncated between amino acids 147 and the C-terminus ofthe natural protein, G332. Variants in which cysteine residues are addeddistal to the final amino acid of a TPO protein that is truncatedbetween amino acids 147 and 332 also are provided.

The novel TPO-derived molecules of this example can be formulated andtested for activity essentially as set forth in Examples 1 and 2,substituting, however, the appropriate assays and other considerationsknown in the art related to the specific proteins of this example.

EXAMPLE 7 Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF)

GM-CSF stimulates the proliferation and differentiation of varioushematopoietic cells, including neutrophil, monocyte, eosinophil,erythroid, and megakaryocyte cell lineages. The amino acid sequence ofhuman GM-CSF (SEQ ID NO: 8) is given in Cantrell et al. (1985) and Leeet al (1985) both incorporated herein by reference.

GM-CSF is produced as a 144 amino acid preprotein that is cleaved toyield a mature 127 amino acid protein. The mature protein has two sitesfor N-linked glycosylation. One site is located at the C-terminal end ofHelix A; the second site is in the A-B loop. GM-CSF has four major alphahelices referred to as helices A-D, helix A encompasses amino acids13-27, helix B encompasses amino acids 55-65, helix C encompasses aminoacids 74-86 and helix D encompasses amino acids 103-116.

This example provides cysteine-added variants at any of the amino acidsthat comprise the N-linked glycosylation sites, i.e., N27C, L28C, S29C,N37C, E38C and T39C. This example also provides cysteine-added variantsin the region proximal to the A helix (amino acids 1-12), distal to theD helix (amino acids 117-127), in the A-B loop (amino acids 28-54), inthe B-C loop (amino acids 66-73), and in the C-D loop (amino acids87-102). This Example also provides cysteine-added variants at the firstthree or last three amino acids in helices A, B, C and D. Preferredsites for introduction of cysteine substitutions in these regions are:A1, P2, A3, R4, S5, P6, S7, P8, S9, T10, Q11, R30, D31, T32, A33, A34,E35, E41, S44, E45, D48, Q50, E51, T53, Q64, G65, R67, G68, S69, L70,T71, K72, K74, G75, T91, E93, T94, S95, A97, T98, T102, I117, D120,E123, V125, Q126 and E127. Variants in which cysteine residues areintroduced proximal to the first amino acid of the mature protein, i.e.,proximal to A1, or distal to the final amino acid in the mature protein,i.e., distal to E127 are provided.

The novel GM-CSF-derived molecules of this example can be formulated andtested for activity essentially as set forth in Examples 1 and 2,substituting, however, the appropriate assays and other considerationsknown in the art related to the specific proteins of this example.

EXAMPLE 8 IL-2

IL-2 is a T cell growth factor that is synthesized by activated T cells.The protein stimulates clonal expansion of activated T cells. Human IL-2is synthesized as a 153 amino acid precursor that is cleaved to yield a133 amino acid mature protein (Tadatsugu et al., 1983; Devos et al.,1983; SEQ ID NO: 9).

The amino acid sequence of IL-2 is set forth in (Tadatsugu et al. 1983;Devos et al. 1983). The mature protein contains three cysteine residues,two of which form a disulfide bond. Cysteine-125 of the mature proteinis not involved in a disulfide bond. Replacement of cysteine-125 withserine yields an IL-2 mutein with full biological activity (Wang et al.,1984). The protein is O-glycosylated at threonine-3 of the matureprotein chain. IL-2 has four major alpha helices referred to as helicesA-D. Helix a encompasses amino acids 1-11, helix B encompasses aminoacids 54-73, helix C encompasses amino acids 83-97 and helix dencompasses amino acids 115-133.

This example provides cysteine-added variants in the last four positionsof the D helix, in the region distal to the D helix (amino acids130-133), in the A-B loop (amino acids 28-53), in the B-C loop (aminoacids 74-82), and in the C-D loop (amino acids 98-114). These variantsare provided in the context of the natural protein sequence or a variantprotein in which the naturally occurring “free” cysteine residue(cysteine-125) has been changed to another amino acid, preferably serineor alanine. Variants in which cysteine residues are introduced proximalto the first amino acid, i.e., A1, or distal to the final amino acid,i.e., T133, of the mature protein, also are provided.

The novel IL-2-derived molecules of this example can be formulated andtested for activity essentially as set forth in Examples 1 and 2,substituting, however, the appropriate assays and other considerationsknown in the art related to the specific proteins of this example.

EXAMPLE 9 IL-3

IL-3 is produced by activated T cells and stimulates the proliferationand differentiation of pleuripotent hematopoietic stem cells. The aminoacid sequence of human IL-3 (SEQ ID NO: 10) is given in Yang et al.(1986); Dorssers et al. (1987) and Otsuka et al. (1988) all incorporatedherein by reference. The protein contains two cysteine residues and twoN-linked glycosylation sites. Two alleles have been described, resultingin isoforms having serine or proline at amino acid position 8 or themature protein.

This example provides cysteine-added variants at any of the amino acidsthat comprise the N-linked glycosylation sites. This example alsoprovides cysteine-added variants in the region proximal to the A helix,distal to the D helix, in the A-B loop, in the B-C loop, and in the C-Dloop. Variants in which cysteine residues are introduced proximal to thefirst amino acid or distal to the final amino acid in the matureprotein, also are provided.

The novel IL-3-derived molecules of this example can be formulated andtested for activity essentially as set forth in Examples 1 and 2,substituting, however, the appropriate assays and other considerationsknown in the art related to the specific proteins of this example.

EXAMPLE 10 IL-4

IL-4 is a pleiotropic cytokine that stimulates the proliferation anddifferentiation of monocytes and T and B cells. IL-4 has been implicatedin the process that leads to B cells secreting IgE, which is believed toplay a role in asthma and atopy. The bioactivity of IL-4 is speciesspecific. IL-4 is synthesized as a 153 amino acid precursor protein thatis cleaved to yield a mature protein of 129 amino acids. The amino acidsequence of human IL-4 (SEQ ID NO:11) is given in Yokota et al. (1986)which is incorporated herein by reference. The protein contains sixcysteine residues and two N-linked glycosylation sites. Theglycosylation sites are located in the A-B and C-D loops.

This example provides cysteine-added variants at any of the amino acidsthat comprise the N-linked glycosylation sites. This example alsoprovides cysteine-added variants in the region proximal to the A helix,distal to the D helix, in the A-B loop, in the B-C loop, and in the C-Dloop. Variants in which cysteine residues are introduced proximal to thefirst amino acid, H1, or distal to the final amino acid, S129, of themature protein are provided.

The novel IL-4-derived molecules of this example can be formulated andtested for activity essentially as set forth in Examples 1 and 2,substituting, however, the appropriate assays and other considerationsknown in the art related to the specific proteins of this example.

EXAMPLE 11 IL-5

IL-5 is a differentiation and activation factor for eosinophils. Theamino acid sequence of human IL-5 (SEQ ID NO: 12) is given in Yokota etal. (1987) which is incorporated herein by reference. The mature proteincontains 115 amino acids and exists in solution as a disulfide-linkedhomodimer. The protein contains both O-linked and N-linked glycosylationsites.

This example provides cysteine-added variants in the region proximal tothe A helix, distal to the D helix, in the A-B loop, in the B-C loop,and in the C-D loop. Variants in which cysteine residues are addedproximal to the first amino acid or distal to the final amino acid inthe mature protein are provided.

The novel IL-5-derived molecules of this example can be formulated andtested for activity essentially as set forth in Examples 1 and 2,substituting, however, the appropriate assays and other considerationsknown in the art related to the specific proteins of this example.

EXAMPLE 12 IL-6

IL-6 stimulates the proliferation and differentiation of many celltypes. The amino acid sequence of human IL-6 (SEQ ID NO: 13) is given inHirano et al. (1986) which is incorporated herein by reference. IL-6 issynthesized as a 212 amino acid preprotein that is cleaved to generate a184 amino acid mature protein. The mature protein contains two sites forN-linked glycosylation and one site for O-glycosylation, at T137, T138,T142 or T143.

This example provides cysteine-added variants at any of the amino acidsthat comprise the N-linked glycosylation sites and at the O-linkedglycosylation site. Also provided are cysteine-added variants in theregion proximal to the A helix, distal to the D helix, in the A-B loop,in the B-C loop, and in the C-D loop. Variants in which cysteineresidues are added proximal to the first amino acid or distal to thefinal amino acid of the mature protein also are provided.

The novel IL-6-derived molecules of this example can be formulated andtested for activity essentially as set forth in Examples 1 and 2,substituting, however, the appropriate assays and other considerationsknown in the art related to the specific proteins of this example.

EXAMPLE 13 IL-7

IL-7 stimulates proliferation of immature B cells and acts on mature Tcells. The amino acid sequence of human IL-7 (SEQ ID NO: 14) is given inGoodwin et al. (1989) which is incorporated herein by reference. Theprotein is synthesized as a 177 amino acid preprotein that is cleaved toyield a 152 amino acid mature protein that contains three sites forN-linked glycosylation.

This example provides cysteine-added variants at any of the amino acidsthat comprise the N-linked glycosylation sites. This example alsoprovides cysteine-added variants in the region proximal to the A helix,distal to the D helix, in the A-B loop, in the B-C loop, and in the C-Dloop.

The novel IL-7-derived molecules of this example can be formulated andtested for activity essentially as set forth in Examples 1 and 2,substituting, however, the appropriate assays and other considerationsknown in the art related to the specific proteins of this example.Variants in which cysteine residues are added proximal to the firstamino acid or distal to the final amino acid of the mature protein alsoare provided.

EXAMPLE 14 IL-9

IL-9 is a pleiotropic cytokine that acts on many cell types in thelymphoid, myeloid and mast cell lineages. IL-9 stimulates theproliferation of activated T cells and cytotoxic T lymphocytes,stimulates proliferation of mast cell precursors and synergizes witherythropoietin in stimulating immature red blood cell precursors. Theamino acid sequence of human IL-9 (SEQ ID NO: 15) is given in Yang etal. (1989) which is incorporated herein by reference. IL-9 issynthesized as a precursor protein of 144 amino acids that is cleaved toyield a mature protein of 126 amino acids. The protein contains fourpotential N-linked glycosylation sites.

This example provides cysteine-added variants at any of the three aminoacids that comprise the N-linked glycosylation sites. This example alsoprovides cysteine-added variants in the region proximal to the A helix,distal to the D helix, in the A-B loop, in the B-C loop, and in the C-Dloop. Variants in which cysteine residues are added proximal to thefirst amino acid or distal to the final amino acid of the mature proteinalso are provided.

The novel IL-9-derived molecules of this example can be formulated andtested for activity essentially as set forth in Examples 1 and 2,substituting, however, the appropriate assays and other considerationsknown in the art related to the specific proteins of this example.

EXAMPLE 15 IL-10

The amino acid sequence of human IL-10 (SEQ ID NO: 16) is given inVieira et al. (1991) which is incorporated herein by reference. IL-10 issynthesized as a 178 amino acid precursor protein that is cleaved toyield a mature protein of 160 amino acids. IL-10 can function toactivate or suppress the immune system. The protein shares structuralhomology with the interferons, i.e., it contains five amphipathichelices. The protein contains one N-linked glycosylation site.

This example provides cysteine-added variants at any of the three aminoacids comprising the N-linked glycosylation site. This example alsoprovides cysteine-added variants in the region proximal to the A helix,distal to the E helix, in the A-B loop, in the B-C loop, in the C-D loopand in the D-E loop. Variants in which cysteine residues are addedproximal to the first amino acid or distal to the final amino acid ofthe mature protein also are provided.

The novel IL-10-derived molecules of this example can be formulated andtested for activity essentially as set forth in Examples 1 and 2,substituting, however, the appropriate assays and other considerationsknown in the art related to the specific proteins of this example.

EXAMPLE 16 IL-11

IL-11 is a pleiotropic cytokine that stimulates hematopoiesis,lymphopoiesis and acute phase responses. IL-11 shares many biologicaleffects with IL-6. The amino acid sequence of human IL-11 (SEQ ID NO:17) is given in Kawashima et al. (1991) and Paul et al. (1990) bothincorporated herein by reference. IL-11 is synthesized as a precursorprotein of 199 amino acids that is cleaved to yield a mature protein of178 amino acids. There are no N-linked glycosylation sites in theprotein. IL-11 has four major alpha helices referred to as helices A-D.Relative to the amino acid sequence shown in SEQ ID NO: 17, helix Aencompasses amino acids 37-56, helix B encompasses amino acids 92-112,helix C encompasses amino acids 125-147 and helix D encompasses aminoacids 173-196. Amino acids 1-21 of SEQ ID NO: 17 encompasses the IL-11signal sequence.

This example provides cysteine-added variants in the region proximal tothe A helix (amino acids 22-36 of SEQ ID NO: 17), distal to the D helix(amino acids 197-199 of SEQ ID NO: 17), in the A-B loop (amino acids57-91 of SEQ ID NO: 17), in the B-C loop (amino acids 113-124 of SEQ IDNO: 17), and in the C-D loop (amino acids 148-172 of SEQ ID NO: 17).This Example also provides cysteine-added variants at the first three orlast three amino acids in helices A, B, C and D. Variants in whichcysteine residues are added proximal to the first amino acid of themature protein (amino acid 22 of SEQ ID NO: 17) or distal to the finalamino acid of the mature protein (amino acid 199 of SEQ ID NO: 17) alsoare provided.

The novel IL-11-derived molecules of this example can be formulated andtested for activity essentially as set forth in Examples 1 and 2,substituting, however, the appropriate assays and other considerationsknown in the art related to the specific proteins of this example.

EXAMPLE 17 IL-12 p35

IL-12 stimulates proliferation and differentiation of NK cells andcytotoxic T lymphocytes. IL-12 exists as a heterodimer of a p35 subunitand a p40 subunit. The p35 subunit is a member of the GH supergenefamily. The amino acid sequence of the p35 subunit (SEQ ID NO: 18) isgiven in Gubler et al. (1991) and Wolf et al. (1991) both incorporatedherein by reference. p35 is synthesized as a precursor protein of 197amino acids and is cleaved to yield a mature protein of 175 amino acids.The protein contains 7 cysteine residues and three potential N-linkedglycosylation sites.

This example provides cysteine-added variants at any of the three aminoacids that comprise the three N-linked glycosylation sites. This examplealso provides cysteine-added variants in the region proximal to the Ahelix, distal to the D helix, in the A-B loop, in the B-C loop, and inthe C-D loop. These variants are provided in the context of the naturalprotein sequence or a variant protein in which the naturally occurring“free” cysteine residue has been changed to another amino acid,preferably serine or alanine. Variants in which cysteine residues areadded proximal to the first amino acid or distal to the final amino acidof the mature protein also are provided.

The novel IL-12 p35-derived molecules of this example can be formulatedand tested for activity essentially as set forth in Examples 1 and 2,substituting, however, the appropriate assays and other considerationsknown in the art related to the specific proteins of this example.

EXAMPLE 18 IL-13

IL-13 shares many biological properties with IL-4. The amino acidsequence of human IL-13 (SEQ ID NO: 19) is given in McKenzie et al.(1993) and Minty et al. (1993) both incorporated herein by reference.The protein is synthesized as a 132 amino acid precursor protein that iscleaved to yield a mature protein of 112 amino acids. The mature proteincontains 5 cysteine residues and multiple N-linked glycosylation sites.A variant in which glutamine at position 78 is deleted due toalternative mRNA splicing has been described (McKenzie et al 1993)

This example provides cysteine-added variants at any of the three aminoacids comprising the N-linked glycosylation sites. This example alsoprovides cysteine-added variants in the region proximal to the A helix,distal to the D helix, in the A-B loop, in the B-C loop, and in the C-Dloop. These variants are provided in the context of the natural proteinsequence or a variant sequence in which the pre-existing “free” cysteinehas been changed to another amino acid, preferably to alanine or serine.Variants in which cysteine residues are added proximal to the firstamino acid or distal to the final amino acid of the mature protein alsoare provided.

The novel IL-13-derived molecules of this example can be formulated andtested for activity essentially as set forth in Examples 1 and 2,substituting, however, the appropriate assays and other considerationsknown in the art related to the specific proteins of this example.

EXAMPLE 19 IL-15

IL-15 stimulates the proliferation and differentiation of T cells, NKcells, LAK cells and Tumor Infiltrating Lymphocytes. IL-15 may be usefulfor treating cancer and viral infections. The sequence of IL-15 (SEQ IDNO: 20) is given in Anderson et al. (1995) which is incorporated hereinby reference. IL-15 contains two N-linked glycosylation sites, which arelocated in the C-D loop and C-terminal end of the D helix. IL-15 encodesa 162 amino acid preprotein that is cleaved to generate a mature 114amino acid protein. IL-15 contains four major alpha helices referred toas helices A-D. Helix A encompasses amino acids 1-15, helix Bencompasses amino acids 38-57, helix C encompasses amino acids 65-78 andhelix D encompasses amino acids 97-114.

This example provides cysteine-added variants at any of the three aminoacids comprising the N-linked glycosylation sites in the C-D loop orC-terminal end of the D helix. This example also provides cysteine-addedvariants proximal to helix A, in the A-B loop (amino acids 16-37), theB-C loop (amino acids 58-64), the C-D loop (amino acids 79-96) or distalto helix D. This Example also provides cysteine-added variants at thefirst three or last three amino acids in helices A, B, C and D. Variantsin which cysteine residues are added proximal to the first amino acid ordistal to the final amino acid of the mature protein also are provided.

The novel IL-15-derived molecules of this example can be formulated andtested for activity essentially as set forth in Examples 1 and 2,substituting, however, the appropriate assays and other considerationsknown in the art related to the specific proteins of this example.

EXAMPLE 20 Macrophage Colony Stimulating Factor (M-CSF)

M-CSF regulates the growth, differentiation and function of monocytes.The protein is a disulfide-linked homodimer. Multiple molecular weightspecies of M-CSF, which arise from differential mRNA splicing, have beendescribed. The amino acid sequence of human M-CSF and its variousprocessed forms are given in Kawasaki et al (1985), Wong et al. (1987)and Cerretti et al (1988) which are incorporated herein by reference.Cysteine-added variants can be produced following the general teachingsof this application and in accordance with the examples set forthherein.

The novel MCSF-derived molecules of this example can be formulated andtested for activity essentially as set forth in Examples 1 and 2,substituting, however, the appropriate assays and other considerationsknown in the art related to the specific proteins of this example.

EXAMPLE 21 Oncostatin M

Oncostatin M is a multifunctional cytokine that affects the growth anddifferentiation of many cell types. The amino acid sequence of humanoncostatin M (SEQ ID NO: 21) is given in Malik et al. (1989) which isincorporated herein by reference. Oncostatin M is produced by activatedmonocytes and T lymphocytes. Oncostatin M is synthesized as a 252 aminoacid preprotein that is cleaved sequentially to yield a 227 amino acidprotein and then a 196 amino acid protein (Linsley et al., 1990). Themature protein contains O-linked glycosylation sites and two N-linkedglycosylation sites. The protein is O-glycosylated at T160, T162 andS165. The mature protein contains five cysteine residues.

This example provides cysteine-added variants at either of the threeamino acids comprising the N-linked glycosylation sites or at the aminoacids that comprise the O-linked glycosylation sites. This example alsoprovides cysteine-added variants proximal to helix A, in the A-B loop,in the B-C loop, in the C-D loop or distal to helix D. These variantsare provided in the context of the natural protein sequence or a variantsequence in which the pre-existing “free” cysteine has been changed toanother amino acid, preferably to alanine or serine. Variants in whichcysteine residues are added proximal to the first amino acid or distalto the final amino acid of the mature protein also are provided.

The novel Oncostatin M-derived molecules of this example can beformulated and tested for activity essentially as set forth in Examples1 and 2, substituting, however, the appropriate assays and otherconsiderations known in the art related to the specific proteins of thisexample.

EXAMPLE 22 Ciliary Neurotrophic Factor (CNTF)

The amino acid sequence of human CNTF (SEQ ID NO: 22) is given in Lam etal. (1991) which is incorporated herein by reference. CNTF is a 200amino acid protein that contains no glycosylation sites or signalsequence for secretion. The protein contains one cysteine residue. CNTFfunctions as a survival factor for nerve cells.

This example provides cysteine-added variants proximal to helix A, inthe A-B loop, the B-C loop, the C-D loop or distal to helix D. Thesevariants are provided in the context of the natural protein sequence ora variant sequence in which the pre-existing “free” cysteine has beenchanged to another amino acid, preferably to alanine or serine. Variantsin which cysteine residues are added proximal to the first amino acid ordistal to the final amino acid of the mature protein also are provided.

The novel CNTF-derived molecules of this example can be formulated andtested for activity essentially as set forth in Examples 1 and 2,substituting, however, the appropriate assays and other considerationsknown in the art related to the specific proteins of this example.

EXAMPLE 23 Leukemia Inhibitory Factor (LIF)

The amino acid sequence of LIF (SEQ ID NO: 23) is given in Moreau et al.(1988) and Gough et al. (1988) both incorporated herein by reference.The human gene encodes a 202 amino acid precursor that is cleaved toyield a mature protein of 180 amino acids. The protein contains sixcysteine residues, all of which participate in disulfide bonds. Theprotein contains multiple O- and N-linked glycosylation sites. Thecrystal structure of the protein was determined by Robinson et al.(1994). The protein affects the growth and differentiation of many celltypes.

This example provides cysteine-added variants at any of the three aminoacids comprising the N-linked glycosylation sites or the O-linkedglycosylation site. Also provided are cysteine-added variants proximalto helix A, in the A-B loop, the B-C loop, the C-D loop or distal tohelix D. Variants in which cysteine residues are added proximal to thefirst amino acid or distal to the final amino acid of the mature proteinalso are provided.

The novel LIF-derived molecules of this example can be formulated andtested for activity essentially as set forth in Examples 1 and 2,substituting, however, the appropriate assays and other considerationsknown in the art related to the specific proteins of this example.

EXAMPLE 24 Expression and Purification of Wild Type Murine GM-CSF andCysteine Muteins of Murine GM-CSF

A. Expression and Purification of Wild Type Murine GM-CSF (muGM-CSF)

Cloning of a cDNA encoding wild type murine GM-CSF (WT muGM-CSF) isdescribed in PCT/US01/16088, incorporated herein by reference in itsentirety. Wild type muGM-CSF was expressed and purified using thefollowing protocol. A fresh saturated overnight culture of E. colistrain W3110 transformed with pBBT257::stII-muGM-CSF, which encodes wildtype muGM-CSF and is described in PCT/US01/16088, was inoculated at˜0.05 OD @ A₆₀₀ in LB containing 10 μg/ml tetracycline. The 400 mlculture was grown in a 2 L baffled shake flask at 28° C. in a gyrotoryshaker water bath at ˜250 rpm. When the culture reached a density of˜0.6 OD, IPTG was added to a final concentration of 0.5 mM. The inducedculture was then incubated overnight for ˜16 h. The cells were pelletedby centrifugation and frozen at −80° C. The cell pellet was thawed andtreated with 5 mL of B-PER™ bacterial protein extraction reagentaccording to the manufacturer's (Pierce Chemical Company) protocols. Theinsoluble portion, and the bulk of the muGM-CSF protein, was recoveredby centrifugation and resuspended in B-PER. This mixture was treatedwith lysozyme (200 μg/mL) for 10 min to further disrupt the bacterialcell walls, and MgCl₂ (10 mM final) and protease-free DNAse (2 μg/ml)were added. Insoluble mGM-CSF was collected by centrifugation andwashed, by resuspension in water and recentrifugation, to remove most ofthe solubilized cell debris. For refolding, the resulting pelletcontaining insoluble muGM-CSF was dissolved in 10 ml of 8 M urea, 25 mMcysteine in 20 mM Tris Base. This mixture was stirred for 30 min at roomtemperature then diluted into 100 ml of 20 mM Tris, 40 μM coppersulfate, 15% glycerol, pH 8.0. This refold mixture was held at 4° C. for2 days and then centrifuged and loaded onto a 5 ml Q-Sepharose HiTrapcolumn (Amersham Pharmacia) equilibrated in 20 mM Tris, pH 8.0 (BufferA). The bound proteins were eluted with a linear salt gradient from0-35% Buffer B (1M NaCl, 20 mM Tris, pH 8). Column fractions wereanalyzed by non-reducing SDS-PAGE. muGM-CSF eluted at approximately 190mM NaCl. Fractions containing primarily muGM-CSF were pooled.

The Q-Sepharose pool was adjusted to 30% ammonium sulfate by slowaddition of solid ammonium sulfate. The mixture was warmed to roomtemperature before being loaded onto a 1 mL Phenyl HP HiTrap column(Amersham Pharmacia) previously equilibrated with 30% ammonium sulfatein 20 mM sodium phosphate, pH 7.5. The muGM-CSF was recovered from thecolumn by elution with a reverse salt gradient (30% ammonium sulfate to0% ammonium sulfate in 20 mM sodium phosphate, pH 7.5). The Phenyl HPcolumn elution profile for GM-CSF showed a single major peak, eluting ata conductivity of approximately 1.28 mS/cm. Column fractions across thepeak were analyzed by non-reducing SDS-PAGE. Fractions containingmuGM-CSF and no visible contaminants were pooled. Protein concentrationswere determined using a MicroBCA kit (Pierce Chemical Company).

B. Expression and Purification of T3C Murine GM-CSF

Construction of the muGM-CSF T3C cysteine mutein (substitution ofcysteine for threonine at position 3 of muGM-CSF) is described inPCT/US01/16088. E. coli strain 3110 transformed withpBBT257::stII-muGM-CSF(T3C), which encodes the muGM-CSF T3C mutein andis described in PCT/US01/16088, was grown, induced and harvested usingthe protocols described in Example 24A for wild type muGM-CSF. Themutein was refolded and purified using the protocol described for wildtype muGM-CSF. The mutein eluted from the Q-Sepharose column atapproximately 220 mM NaCl and from the Phenyl HP column at aconductivity around 2 mS/CM. The mutein was recovered predominantly as amonomer, with an apparent molecular weight of approximately 14 kDa bynon-reducing SDS-PAGE. Protein concentrations were determined using aMicroBCA kit (Pierce Chemical Company).

EXAMPLE 25 PEGylation of the muGM-CSF T3C Mutein

One milligram of muGM-CSF T3C protein prepared as described in Example24B was diluted 3-fold with 100 mM Tris, pH 8, to a final concentrationof 0.1 mg/ml protein. A 20-fold molar excess of 20 kDamaleimide-Polyethylene Glycol (20 kDa-mPEG-maleimide, ShearwaterCorporation) was added followed by a 15× fold excess of TCEP (PierceChemical Company). The mixture was allowed to sit at room temperaturefor 4 hours before being diluted 5-fold with 20 mM Tris pH 8. ThePEGylation reaction was next loaded on to a 1 ml Q-Sepharose HiTrapcolumn (Amersham Pharmacia) equilibrated in 20 mM Tris, pH 8.0 (BufferA). The bound proteins were eluted with a linear salt gradient from0-35% Buffer B (1M NaCl, 20 mM Tris, pH 8). Column fractions wereanalyzed by non-reducing SDS-PAGE. The T3C protein modified with the 20kDa maleimide-PEG eluted at approximately 90 mM NaCl. Fractionscontaining purified 20 kDa PEG-T3C protein were pooled. The T3C proteinmodified with a 10 kDa mPEG-maleimide (Shearwater Corporation) and a 40kDa mPEG2 maleimide (Shearwater Corporation) were prepared using thissame protocol.

EXAMPLE 26 Effects of Wild Type muGM-CSF and PEG-T3C GM-CSF Proteins onHematopoiesis

The purpose of this study was to determine if the PEG-T3C GM-CSFproteins are effective at stimulating hematopoiesis in a mammal. Thestudy was performed in rats because rats are predictive of the situationin other mammals, including humans. Similar types of experiments can beperformed with other PEGylated GM-CSF cysteine muteins such as thosedescribed in Example 7 and in PCT/US01/16088 to demonstrate that theseproteins are effective at stimulating hematopoiesis. The PEGylatedGM-CSF cysteine muteins will be effective from the low μg/kg to themg/kg range with the preferred level being between 25-300 μg/kg. MaleSprague Dawley rats weighing approximately 350 grams were given a singleintravenous dose of wild type muGM-CSF, or the T3C mutein modified with10 kDa-, 20 kDa and 40 kDa-maleimide PEGs. All rats were dosed at 100μg/kg. The protein solutions were prepared at 100 μg/ml in phosphatebuffered saline. Blood samples (0.4 ml) were collected at pre-dose, 0.25h, 1.5 h, 4 h, 10 hr, 24 h, 48 h, 72 hr, 96 h, 120 h and 144 hpost-injection in an EDTA-coated tube. Blood samples were centrifugedand the plasma stored at −80° C. At pre-dose, 4, 10, 24, 48, 72, 96, 120and 144 h post-injection approximately 0.2 ml of whole EDTA blood wasremoved prior to centrifugation and placed in an EDTA microtainer tubefor submission to a commercial firm for a complete blood cell (CBC)analysis. Results of the CBC analyses are shown in Tables 1-5. The datashow that the PEG-T3C GM-CSF proteins are effective at stimulatinghematopoiesis in a mammal. In particular the proteins are effective atincreasing blood levels of white blood cells, granulocytes (neutrophilsand eosinophils), monocytes and lymphocytes. TABLE 1 Effects of wildtype muGM-CSF and 10 kDa-, 20 kDa- and 40 kDa-PEG T3C muGM-CSF on whiteblood cell counts 10 kDa-PEG T3C 20 kDa-PEG T3C 40 kDa-PEG T3C Wild typemuGM-CSF (thousand cells/μl) (thousand cells/μl) (thousand cells/μl)(thousand cells/μl) Time (h) Rat 1 Rat 2 Rat 3 Rat 4 Rat 5 Rat 6 Rat 7Rat 8 Rat 9 Rat 10 Rat 11 Rat 12 0 9.2 9.0 8 7.8 7.7 4.3 3.5 3.7 6.6 3.16 2.4 4 19 11 11 13.8 8.8 14 12 7.4 11 11.3 17 10.7 10 12.5 12.7 11.814.6 12.3 14.1 15.9 9 12.5 10.5 17.5 10.2 24 8.2 9.2 8.1 15.2 11.4 9.516 8.7 15 6 8.7 5.8 48 8.9 9.6 8.4 10.9 11.5 8.9 27.9 13.3 17.1 5.7 9.16.4 72 9.2 10.9 8.3 8.7 9.6 8.8 17.8 11.2 17.7 5 13.1 7.3 96 12 11.6 9.213.1 14 14.1 22.6 14.4 20.1 8.4 11.9 11.2 120 9.2 8.9 7.2 10.4 11.5 11.816 14 13.8 9 10.3 14.9 144 5.3 6 3.3 4.5 2.2 4.8 4.8 8.2 4.7 6 6.6 7.1

TABLE 2 Effects of wild type muGM-CSF and 10 kDa-, 20 kDa- and 40kDa-PEG T3C muGM-CSF on blood neutrophil counts 10 kDa-PEG T3C 20kDa-PEG T3C 40 kDa-PEG T3C Wild type muGM-CSF (cells/μl) (cells/μl)(cells/μl) (cells/μl) Time (h) Rat 1 Rat 2 Rat 3 Rat 4 Rat 5 Rat 6 Rat 7Rat 8 Rat 9 Rat 10 Rat 11 Rat 12 0 1104 1350 1360  624  770  344  350 407  660  186  600  96 4 13300 5280 6050 9522 4644 9240 6240 5254 56106102 13600  8346 10 5625 5969 5546 10658  7134 9447 9815 6300 8250 43057875 5918 24 2378 1564 2106 7752 5928 3895 9280 6177 10650  1620 19142088 48 1157 1344 1680 3052 3450 1869 13392  7315 9234 1026 1820 1984 72552  981 1079 1044  960  528 2492 4032 5487  450 1965 2628 96 1320 15081380 2882 2660 2679 5650 5904 5829 1428 2142 4368 120 828 1246 1008 17681265 1652 2400 5880 2622 1980 1339 8344 144 530 1020  132 1260  704 1008 960 3444  987 1260 1188 4118

TABLE 3 Effects of wild type muGM-CSF and 10 kDa-, 20 kDa- and 40kDa-PEG T3C muGM-CSF on blood monocyte counts 10 kDa-PEG T3C 20 kDa-PEGT3C 40 kDa-PEG T3C Wild type muGM-CSF (cells/μl) (cells/μl) (cells/μl)(cells/μl) Time (h) Rat 1 Rat 2 Rat 3 Rat 4 Rat 5 Rat 6 Rat 7 Rat 8 Rat9 Rat 10 Rat 11 Rat 12 0 460 360 400 390 154  86  35  37  66  31 120  04 950 330 440 276 352 280 120 148 220 339 340 214 10 250 508 354 292 492282 151  90 125 210 350 102 24 246 184 162 1S2 228 190 160  87 150 180174  58 48 356 384 336 436 460 3S6 558 399 342 171 273 192 72  92 218166  87  96  88 178 224 354  50 262 146 96 240 232 276 393 280 282 678432 804 168 238 448 120  82 178 144 208 345 354 800 700 276 360 206 447144 106 240  33  45  44  48  96 164  94  60  66 142

TABLE 4 Effects of wild type muGM-CSF and 10 kDa-, 20 kDa- and 40kDa-PEG T3C muGM-CSF on blood eosinophil counts 10 kDa-PEG T3C 20kDa-PEG T3C 40 kDa-PEG T3C Wild type muGM-CSF (cells/μl) (cells/μl)(cells/μl) (cells/μl) Time (h) Rat 1 Rat 2 Rat 3 Rat 4 Rat 5 Rat 6 Rat 7Rat 8 Rat 9 Rat 10 Rat 11 Rat 12 0 92 90 80 156 77 45 35 37 66 31 60 244 0 110 110 138 88 280 120 0 110 113 0 0 10 250 254 236 292 123 141 15190 125 210 175 102 24 492 184 405 760 342 285 480 174 150 240 87 58 48178 96 252 545 345 267 837 266 342 171 91 64 72 92 109 249 261 192 88356 112 354 300 131 73 96 240 116 184 262 140 141 226 288 201 168 119225 120 184 178 216 208 230 236 320 280 138 180 103 149 144 0 120 0 9044 96 96 164 47 120 66 142

TABLE 5 Effects of wild type muGM-CSF and 10 kDa-, 20 kDa- and 40kDa-PEG T3C muGM-CSF on blood lymphocyte counts 10 kDa-PEG T3C 20kDa-PEG T3C 40 kDa-PEG T3C Wild type muGM-CSF (cells/μl) (cells/μl)(cells/μl) (cells/μl) Time (h) Rat 1 Rat 2 Rat 3 Rat 4 Rat 5 Rat 6 Rat 7Rat 8 Rat 9 Rat 10 Rat 11 Rat 12 0 7544 7200 6160 6630 6699 3827 30803219 5808 2852 5220 2280 4 4750 5280 4400 3864 3696 4200 5520 1998 50604746 3060 2140 10 6375 5969 5664 3358 4551 4230 4983 2520 4000 5775 91004080 24 5084 7268 5427 6536 4902 5130 6080 2262 4050 3960 6525 3596 487209 7776 6132 6867 7245 6408 13113  5320 7182 4332 6916 4160 72 84649592 6806 7306 8352 8096 14774  6832 11505  4200 10742  4453 96 10200 9976 7360 9563 10920  10998  16046  7776 13266  6636 9401 6160 120 80967298 5832 8216 9660 9558 12480  7140 10764  6480 8652 5960 144 4664 46203135 3105 1408 3648 3648 4428 3572 4560 5280 2698

EXAMPLE 27 Accelerating Recovery from Neutropenia with PEGylated GM-CSFCysteine Mutein

The purpose of this study was to determine whether the PEGylated GM-CSFcysteine muteins could accelerate recovery from chemotherapy-inducedneutropenia in a mammal. The study was performed in rats because ratsare predictive of neutropenia in other mammals, including humans.Similar types of experiments can be performed with other PEGylatedGM-CSF cysteine muteins such as those described in Example 7 and inPCT/US01/16088 to demonstrate that these proteins are effective ataccelerating recovery from neutropenia. The PEGylated GM-CSF muteinswill be effective from the low μg/kg (e.g., at least about 0.1 μg/kg) tothe mg/kg range (e.g., at least about 0.1 mg/kg) with the preferredlevel being between 25-300 μg/kg.

Male Sprague Dawley rats (5 animals per group) weighing approximately180 g were given an intraperitoneal dose of cyclophosphamide (CPA; 100mg/kg) on Day 0. Wild type muGM-CSF, 20 kDa-PEG-T3C muGM-CSF or 40kDa-PEG-T3C muGM-CSF prepared as described in Example 25 were theninjected subcutaneously into rats at a dose of 100 μg/kg on Days 1-5.Protein samples were prepared in phosphate buffered saline. As controls,different groups of rats were injected with wild type muGM-CSF (Example24) or vehicle (phosphate buffered saline). There was one group of ratsthat received no chemotherapy. A blood sample was collected at pre-dose(Day 0) and on Days 1-11 and 13 from all groups to perform a completeblood cell count (CBC) analysis. 400 μl of whole EDTA blood was removedand placed in an eppendorf tube for submission to an external lab forCBC analysis.

Results of the CBC analysis for white blood cells and neutrophils areshown in Tables 6 and 7. CPA-treatment caused a drastic drop inneutrophil and white blood cell counts by Day 1. It was found that the20 kDa-PEG-T3C and 40 kDa-PEG-T3C muGM-CSF accelerated the recovery ofneutropenia compared to the CPA-only treated group and the wild typemuGM-CSF treated group. Neutrophil counts returned to normal levels (day0 values) between Days 5 and 6 for the PEG-T3C muGM-CSF proteins versusbetween Day 9 and 10 for the CPA-only treated animals and the wild typemuGM-CSF treated animals. White blood cell counts returned to normallevels between Days 6 and 8 for the PEG-T3C muGM-CSF proteins, whereaswhite blood cell counts in the CPA-treated animals and the wild typemuGM-CSF treated animals had not returned to normal levels (day 0values) even by day 13 when the experiment was terminated. Similar typesof experiments could be performed using different dosing schedules,e.g., a single injection on day 1, every other day injections or everythird day injections, or weekly injections to determine an optimumdosing schedule for the PEG-GM-CSF cysteine muteins. TABLE 6 Effects ofmultiple doses of muGM-CSF, 20 kDa-PEG-T3C and 40 kDa-PEG-T3C on whiteblood cell (WBC) counts in neutropenic rats White Blood Cells (1000/μL)*Time Control (Day) (Vehicle) CPA-alone muGM-CSF 20 kDa-PEG-T3C 40kDa-PEG-T3C 0  9.8 +/− 0.5 9.4 +/− 0.5 9.8 +/− 0.5 9.8 +/− 0.8 10.4 +/−0.3  1  9.1 +/− 0.2 3.4 +/− 0.5 3.3 +/− 0.2 3.1 +/− 0.2 3.3 +/− 0.3 213.6 +/− 0.5 2.2 +/− 0.2 1.6 +/− 0.2 1.4 +/− 0.1 2.3 +/− 0.3 3 11.2 +/−0.5 1.6 +/− 0.6 1.2 =/− 0.3 4.3 +/− 0.5 3.7 +/− 0.4 4 13.6 +/− 0.5 2.3+/− 0.2 1.6 +/− 0.2 1.4 +/− 0.1 2.3 +/− 0.3 5 11.2 +/− 1.0 1.0 +/− 0.11.0 +/− 0.1 0.9 +/− 0.1 1.3 +/− 0.4 6 10.4 +/− 0.4 1.4 +/− 0.3 1.4 +/−0.1 4.3 +/− 0.6 4.0 +/− 1.1 7 11.6 +/− 0.6 2.2 +/− 0.3 3.4 +/− 0.3 11.1+/− 1.1  8.7 +/− 1.4 8 11.8 +/− 0.5 3.0 +/− 0.2 3.8 +/− 0.3 10.6 +/−0.7  11.4 +/− 1.4  9 14.6 +/− 0.4 3.6 +/− 0.2 4.9 +/− 0.4 10.1 +/− 1.0 12.3 +/− 2.1  10 11.3 +/− 0.8 4.8 +/− 0.5 6.5 +/− 0.3 7.2 +/− 0.6 9.6+/− 1.9 11 11.1 +/− 0.9 5.0 +/− 0.3 6.5 +/− 0.2 5.7 +/− 0.4 6.2 +/− 0.913 13.2 +/− 0.4 6.6 +/− 0.5 6.2 +/− 0.3 5.0 +/− 0.8 5.1 +/− 0.2*WBC counts are means ± SE from 5 rats/group

TABLE 7 Effects of multiple doses of muGM-CSF, 20 kDa-PEG-T3C and 40kDa-PEG-T3C on absolute neutrophil counts in neutropenic rats AbsoluteNeutrophil Count (cells/μL)* Time Control (Day) (Vehicle) CPA-alonemuGM-CSF 20 kDa-PEG-T3C 40 kDa-PEG-T3C 0 1333 +/− 90  1352 +/− 153 1328+/− 111 1330 +/− 177 1335 +/− 103 1 1778 +/− 121  902 +/− 191 700 +/− 38735 +/− 78 790 +/− 68 2 1659 +/− 280 602 +/− 82 154 +/− 14 566 +/− 641181 +/− 217 3 1688 +/− 233  428 +/− 193 277 +/− 91 3060 +/− 333 2620+/− 238 4 1659 +/− 280  625 +/− 101 154 +/− 14 566 +/− 64 1181 +/− 217 51108 +/− 187 130 +/− 66 152 +/− 41 236 +/− 57  370 +/− 155 6 1355 +/−222 103 +/− 35 189 +/− 49 2041 +/− 369 2215 +/− 844 7 1445 +/− 196 227+/− 53  593 +/− 136 6366 +/− 759 4338 +/− 999 8 1786 +/− 240 338 +/− 35 631 +/− 220 4870 +/− 321 5171 +/− 836 9 2371 +/− 221  676 +/− 161 1156+/− 197 3277 +/− 436  3636 +/− 1346 10 2178 +/− 279 1617 +/− 228 2573+/− 135 2508 +/− 332 2636 +/− 887 11 1964 +/− 164 2337 +/− 183 2952 +/−360 2296 +/− 304 2161 +/− 313 13 1830 +/− 159 2244 +/− 166 1843 +/− 1721502 +/− 222 1433 +/− 72 *Neutrophil counts are means ± SE for 5 rats/group

All of the documents cited herein are incorporated herein by reference.

The protein analogues disclosed herein can be used for the knowntherapeutic uses of the native proteins in essentially the same formsand doses all well known in the art.

While the exemplary preferred embodiments of the present invention aredescribed herein with particularity, those having ordinary skill in theart will recognize changes, modifications, additions, and applicationsother than those specifically described herein, and may adapt thepreferred embodiments and methods without departing from the spirit ofthis invention.

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1. A method to protect an animal from a disease or condition that can betreated by wild-type granulocyte-macrophage colony stimulating factor(GM-CSF), comprising administering to an animal having said disease orcondition a composition comprising a granulocyte-macrophage colonystimulating factor (GM-CSF) cysteine mutein.
 2. The method of claim 1,wherein said GM-CSF cysteine mutein comprises at least one non-naturalcysteine residue which has been added, either by substitution for anamino acid in the natural protein sequence or by insertion between twoadjacent amino acids in the natural protein sequence, in a region of theprotein selected from the group consisting of: the A-B loop, the B-Cloop, the C-D loop, the area near the ends of helix A, the area near theends of helix B, the area near the ends of helix C, the area near theends of helix D, the region proximal to helix A, and the region distalto helix D.
 3. The method of claim 1, wherein said GM-CSF cysteinemutein comprises at least one non-natural cysteine residue which hasbeen added preceding the N-terminal amino acid of the mature protein orfollowing the C-terminal amino acid of the protein.
 4. The method ofclaim 1, wherein said GM-CSF cysteine mutein is a GM-CSF proteincomprising a substitution of cysteine for the amino acid at position 3of wild-type GM-CSF.
 5. The method of claim 1, wherein said GM-CSFcysteine mutein is a human GM-CSF cysteine mutein.
 6. The method ofclaim 5, wherein said cysteine mutein comprises at least one cysteineresidue substituted for an amino acid in wild-type GM-CSF (SEQ ID NO:8)selected from the group consisting of: A1, P2, A3, R4, S5, P6, S7, P8,S9, T10, Q11, R30, D31, T32, A33, A34, E35, E41, S44, E45, D48, Q50,E51, T53, Q64, G65, R67, G68, S69, L70, T71, K72, K74, G75, T91, E93,T94, S95, A97, T98, T102, I117, D120, E123, V125, Q126 and E127.
 7. Themethod of claim 5, wherein said cysteine mutein comprises at least onecysteine residue substituted for the having a substitution of cysteinefor alanine at position 3 of human GM-CSF (SEQ ID NO:8).
 8. The methodof claim 5, wherein said cysteine mutein comprises at least one cysteineresidue substituted for the having a substitution of cysteine foralanine at position 1 of human GM-CSF (SEQ ID NO:8).
 9. The method ofclaim 5, wherein said cysteine mutein comprises at least one cysteineresidue substituted for the having a substitution of cysteine for serineat position 5 of human GM-CSF (SEQ ID NO:8).
 10. The method of claim 5,wherein said cysteine mutein comprises at least one cysteine residuesubstituted for the having a substitution of cysteine for serine atposition 7 of human GM-CSF (SEQ ID NO:8). 11-15. (canceled)
 16. A methodto prevent or treat the occurrence of neutropenia in an animalcomprising administering to said animal a composition comprising agranulocyte-macrophage colony stimulating factor (GM-CSF) cysteinemutein.
 17. (canceled)
 18. (canceled)
 19. The method of claim 16,wherein said neutropenia is selected from the group consisting of: a)neutropenia resulting from myelosuppressive chemotherapy; b) neutropeniaassociated with bone marrow transplantation; c) neutropenia associatedwith infection with the human immunodeficiency virus; d) neutropeniaassociated with burns, surgery, dilatation, anemia and neonatalsepticemia; e) severe chronic neutropenia; f) neutropenia associatedwith aplastic anemia and acute leukemia.
 20. The method of claim 16,wherein said neutropenia is neutropenia resulting from myelosuppressivechemotherapy.
 21. The method of claim 16, wherein said neutropenia isneutropenia associated with bone marrow transplantation.
 22. The methodof claim 16, wherein said neutropenia is neutropenia associated withinfection with the human immunodeficiency virus.
 23. The method of claim16, wherein said neutropenia is neutropenia associated with burns,surgery, dilatation, anemia and neonatal septicemia.
 24. The method ofclaim 16, wherein said neutropenia is severe chronic neutropenia. 25.The method of claim 16, wherein said neutropenia is neutropeniaassociated with aplastic anemia and acute leukemia. 26-34. (canceled)35. A method for stimulating the expansion and proliferation ofperipheral blood progenitor cells in an animal comprising administeringto said animal a composition comprising a granulocyte-macrophage colonystimulating factor (GM-CSF) cysteine mutein. 36-40. (canceled)
 41. Acysteine variant of interleukin-11 of SEQ ID NO: 17, wherein a cysteineresidue is substituted for at least one amino acid located in at leastone region of interleukin-11 selected from the group consisting of: theA-B loop (amino acids 57-91 of SEQ ID NO: 17), the B-C loop (amino acids113-124 of SEQ ID NO: 17), the C-D loop (amino acids 148-172 of SEQ IDNO: 17), the first three or last three amino acids in helix A, the firstthree or last three amino acids in helix B, the first three or lastthree amino acids in helix C, the first three or last three amino acidsin helix D, the amino acids preceding helix A (amino acids 22-36 of SEQID NO: 17), and the amino acids following helix D (amino acids 197-199of SEQ ID NO: 17); wherein said variant stimulates in vitroproliferation of a cell line that proliferates in response tointerleukin-11.